E. coli strain GB08-red大肠杆菌菌株 BioVector NTCC质粒载体菌种细胞基因保藏中心

E. coli strain GB08-red大肠杆菌菌株

Description
Recombineering-proficient E. coli strain for linear plus circular homologous recombination.
Features
Recombineering-proficient E. coli strain
E. coli strain harboring an arabinose inducible γβαA operon (redγ, redβ, redα and recA) at the ybcC locus
Applications
All types of "linear plus circular" homologous recombination experiments, e.g. plasmid modifications
Description
E.coli strain GB08-red enables recombineering of plasmids and BACs without the necessity to maintain an expression plasmid in the cells since the necessary genes are located on the chromosome.

Introduction
Red/ET recombination relies on homologous recombination in vivo in E.coli and
allows a wide range of modifications on DNA molecules of any size and at any
chosen position. Homologous recombination is the exchange of genetic material
between two DNA molecules in a precise, specific and accurate manner.
Homologous recombination occurs through homology regions, which are stretches of
DNA shared by the two molecules that recombine. Because the sequence of the
homology regions can be chosen freely, any position on a target molecule can be
specifically altered. Red/ET recombination allows you to choose homology arms as
short as 50 bp for homologous recombination, which can easily be added to a
functional cassette by long PCR primers.
E. coli strain GB08-red harbours an arabinose inducible gbaA operon (redγ, redβ,
redα and recA) at the ybcC locus (Fu et al. 2010).
The “PGK-gb2-neo” cassette supplied with the kit is designed to allow
kanamycin/neomycin selection in prokaryotic and eukaryotic cells, respectively. It
combines a prokaryotic promoter (“gb2”) for expression of kanamycin resistance in
E.coli with a eukaryotic promoter (“PGK”) for expression of neomycin resistance in
mammalian cells. The prokaryotic promoter gb2 is a slightly modified version of the
Em7 promoter; it mediates higher transcription efficiency than the generally used Tn5
promoter. The promoter of the mouse phosphoglycerate kinase gene (PGK) is used
as the eukaryotic promoter. A synthetic polyadenylation signal terminates
kanamycin/neomycin expression.

Experimental Outline

Figure 1: workflow.
1. Transform E. coli GB08-red cells with a mixture of linear DNA and
circular plasmid. For your convenience a plasmid and a control insert
(kanamycin resistance marker gene) sharing 50bp of terminal homology arms
are provided for a control experiment.
2. Red/ET Recombination step. The expression of redα and redβ genes
mediating Red/ET recombination is induced by the addition of L-arabinose.
After induction, the cells are prepared for electroporation. Subsequently the
circular plasmid and the linear fragment (PCR product) are co-electroporated.
Since the PCR product encodes for a kanamycin resistance gene only
colonies carrying successfully modified plasmids will survive kanamycin
selection on the agar plates. Subsequent DNA mini preparation is used to
confirm the successful integration of the DNA fragment.
In most cases an additional retransformation step is required to isolate the
modified plasmid from all copies of the original plasmid.

3 How Red/ET Recombination works
Target DNA molecules are precisely altered by homologous recombination in E. coli
cells which express the phage-derived protein pairs, either RecE/RecT from the Rac
prophage, or Redα/Redβ from λ phage. These protein pairs are functionally and
operationally similar. RecE and Redα are 5‘- 3‘ exonucleases, and RecT and Redβ
are DNA annealing proteins. A functional interaction between RecE and RecT, or
between Redα and Redβ is also required in order to catalyse the homologous
recombination reaction. Recombination occurs through homology regions, which are
stretches of DNA shared by the two molecules that recombine (Figure 2). The
recombination is further assisted by λ-encoded Gam protein, which inhibits the
RecBCD exonuclease activity of E.coli.


Double-stranded break repair (DSBR) is initiated by the recombinase protein pairs,
RecE/RecT or Redα/Redβ.
First RecE (or Redα) digests one strand of the DNA from the double-stranded break,
leaving the other strand as a 3’ ended, single-stranded DNA overhang. Then RecT
(or Redβ) binds and coats the single strand. The protein-nucleic acid filament aligns
with homologous DNA. Once aligned, the 3’ end becomes a primer for DNA
replication.
E. coli strain GB08-red harbours an arabinose inducible gbaA operon (redγ, redβ,
redα and recA) at the ybcC locus (Fu et al. 2010). The recombination window is
therefore limited by the transient expression of the recombineering proteins. Thus,
the risk of unwanted intra-molecular rearrangement is minimized.
4 Technical protocol
4.1 Generation of a functional cassette flanked by homology arms
Oligonucleotide design
I) Choose 50 nucleotides (N)50 directly adjacent upstream (5’) to the intended
insertion site. Order an oligonucleotide with this sequence at the 5’ end. At the 3’ end
of this oligo include the PCR primer sequence for amplification of the PGK-gb2-neo
cassette, given in italics below.
Upper oligonucleotide (oligo 1): 5’-(N)50 *AATTAACCCTCACTAAAGGGCG -3’
II) Choose 50 nucleotides (N)50 directly adjacent downstream (3’) to the intended
insertion site and transfer them into the reverse complement orientation. Order an
oligonucleotide with this sequence at the 5’ end. At the 3’ end of this oligo, include
the 3’ PCR primer sequence (also in reverse complement orientation) for the PGK gb2-neo cassette, given in italics below.
Lower oligonucleotide (oligo 2): 5’-(N)50 *TAATACGACTCACTATAGGGCTC -3’
If desired, include restriction sites or other short sequences in the ordered oligo(s)
between the 5’ homology regions and the 3’ PCR primer sequences (*).
PCR
The oligonucleotides are suspended in dH2O at a final concentration of 10 pmol/µl. A
standard PCR protocol is given below.
PCR reaction (in 50 µl)
38.5 µl dH2O
5.0 µl 10 x PCR reaction buffer
2.0 µl 5 mM dNTP
1.0 µl upper oligonucleotide
1.0 µl lower oligonucleotide
1.0 µl PCR-template
0.5 µl Taq polymerase (5 U/µl)
• An annealing temperature of 57°- 62°C is optimal.
• Thirty cycles; 1’ 95°; 1’ 57°-62° C; 2.5’ 72° C
4.2 Insertion of a PGK-gb2-neo cassette into a plasmid backbone
In the next step, prepare electro-competent cells from strain GB08-red, shortly after
inducing the expression of the recombination proteins.
In advance, prepare the linear DNA fragment with homology arms that you will insert
into your plasmid. Use tube 2 (PGK-gb2-neo PCR-product) and tube 3 (pSub-Hoxa11)
to perform a control experiment in parallel.
1. Inoculate 1.0 ml LB medium without addition of antibiotics with GB08-red and
incubate the culture over night at 37°C.
2. Before starting the next day:
• Chill ddH2O (or 10% glycerol) on ice for at least 2 h.
• Chill electroporation cuvettes (1 mm gap).
• Cool benchtop centrifuge to 2°C.
Set up 4 lid-punctured microfuge tubes (2 for your own experiment and 2 for
control experiment) containing 1.4 ml each of fresh LB medium and inoculate
two of them with 30 µl fresh overnight culture for your experiment, the other two
with 30 µl of the overnight culture from the control experiment. Incubate the
tubes at 37°C for 1,5 h shaking at 1100 rpm until O D600 ~ 0.3.
3. Add 50 µl 10% L-arabinose to one of the tubes for your own experiment and to
one of the control tubes, giving a final concentration of 0.3%-0.4%. This will
induce the expression of the Red/ET Recombination proteins. Do not use D arabinose. Leave the other tubes without induction as negative controls.
Incubate all at 37°C, shaking for 35 minutes.
7 BioVector NTCC Inc. – Recombineering-proficient E.coli Strain GB08-red, Version 1.0 (October 2012)
Remark: Prepare a 10% L-arabinose (Sigma A-3256) stock solution (in
ddH2O), and use it fresh or frozen in small aliquots at -20°C. Frozen aliquots
should not undergo more than three freeze-thaw cycles.
4. Prepare the cells for electroporation: Centrifuge for 30 sec at 11,000 rpm in a
cooled microfuge benchtop centrifuge (at 2°C). Disc ard the supernatant by
quickly tipping it out twice, and place the pellet on ice. Resuspend the pellet
with 1 ml chilled ddH2O (or 10% glycerol), pipetting up and down three times
to mix the suspension. Repeat the centrifugation and resuspend the cells
again. Centrifuge and tip out the supernatant once more; 20 to 30 µl will be left
in the tube with the pellet. Keep the tube on ice.
5. Add 100 ng of the linear fragment and 100 ng of the circular plasmid to each
of the two microfuge tubes (induced and uninduced), and pipette the mixture
into the chilled electroporation cuvette. In parallel, pipette 1 µl from tube 3 and
4 into each of the two tubes of the control.
6. Electroporate at 1350 V, 10 µF, 600 Ohms. This setting applies to an
Eppendorf®
Electroporator 2510 using an electroporation cuvette with a slit of
1 mm. Other devices can be used, but 1350 V and a 5 ms pulse are
recommended.
7. Add 1 ml LB medium without antibiotics to the cuvette. Mix the cells carefully
by pipetting up and down and pipette back into the microfuge tube. Incubate
the cultures at 37°C with shaking for about 90 min. Recombination will now
occur.
8. Streak the cultures with a loop onto LB agar plates containing the appropriate
antibiotics for the plasmid [e.g. ampicillin (100 µg/ml) plus kanamycin (50
µg/ml) for the control]. You should obtain >100 colonies and the ratio of
induced : uninduced bacterial colonies should exceed 10:1.
More than 95% of all colonies growing on the agar plates conditioned with the
appropriate antibiotics will have successfully recombined copies of the plasmid. Please
note that although most kanamycin-resistant colonies will contain the correct plasmid
recombinant, in rare cases it is possible that secondary recombination, usually deletions
between internal repeats in the plasmid, can also occur.
To confirm the correct recombination event, pick 10 – 20 colonies from your experiment
and 2 from the control reaction, isolate plasmid DNA and analyze the DNA by restriction
digestion.
8
4.3 Verification of successfully modified plasmid by restriction
analysis
Analyze an aliquot of your plasmid DNA by restriction digestion. For the control
experiment, the restriction pattern for the original plasmid pSub-Hoxa11 after BglI digest
is 422 bp, 692 bp, 1730 bp, 1836 bp, 1959 bp, 3485 bp and 7759 bp. The integration of
the PGK-gb2-neo cassette leaves the smaller fragments intact but results in a cleavage
of the 7759 bp fragment into two smaller ones with 4162 bp and 5234 bp respectively.


Figure 3: Restriction analysis of pSub-Hoxa11 (lanes 1-2) and 10 clones after insertion
of the PGK-gb2-neo cassette (lanes 3-12) after BglI digestion. M: Hyperladder I
(Bioline).
Although nearly all clones will show the expected restriction pattern for a successful
integration of the PGK-gb2-neo cassette, the mother plasmid usually still persists in
the cell. High copy plasmids like pBluescript or pSub11-Hoxa, which is used for the
control experiment, replicate to up to several hundred copies per cell. Due to this
high copy number, not all plasmid copies will be recombined at the same time
resulting in a mixed “phenotype” where both plasmids are detectable side by side in
the cell (see also Figure 3 for the control experiment).
To separate the modified plasmid from its unmodified mother plasmid, take a small
amount of the isolated plasmid DNA (about 1 ng) and re-transform a fresh aliquot of
competent E. coli cells with it. Pick several colonies the next day, perform plasmid
mini-prep plasmid DNA isolation following the protocol of your choice and check these
DNA preparations by restriction digestion.

After the re-transformation step the majority of the analyzed clones should show the
restriction pattern for the modified plasmid only. For the control experiment, the
restriction pattern for the plasmid pSub-Hoxa11-neo after BglI digest is 422 bp, 692 bp,
1730 bp, 1836 bp, 1959 bp, 3485 bp, 4162 bp and 5234 bp (Figure 4).


Figure 4: Restriction analysis (BglI digestion) of four colonies obtained in the control
experiment after re-transformation. M: Hyperladder I (Bioline). While clone 1 still
contains both plasmids in the cell as indicated by the 7759 bp fragment, the three other
clones show only the pattern expected for pSub-Hoxa11-neo.
4.4 Maps and sequences
Figure 5: Diagram of the arabinose inducible red operon (redγβα and recA) inserted
at the ybcC locus.

5 Troubleshooting
Problems with the recombination reaction can be caused by a number of different
factors. Please review the information below to troubleshoot your experiments.
We highly recommend performing a positive control experiment using the
components provided in the kit.
For homologous recombination the two DNA molecules must share two regions of
perfect sequence identity. Wrong nucleotides in the homology region can completely
abolish recombination. Since oligonucleotides are used to add the homology regions
they have to be synthesized properly and be of excellent quality. Take into account
that long oligonucleotides (especially if they are longer than 80bp) require additional
purification steps, such as HPLC. Also note that the electronic sequences provided
from databases may not be 100% correct.
If you are trying to target a repeated sequence in your BAC or plasmid, you may
experience problems because the homology region at the end of the linear fragment
can target more than one site.
Observation:

No colonies on your plate after Red/ET Recombination:
If you do not obtain any colonies after recombination, the following parameters
should be checked:
1) The linear DNA fragments/PCR products and the circular plasmid:
o could be wrong (check it by restriction digest or sequencing)
o could be degraded (check an aliquot on an agarose gel)
o could have incorrect homology arms (if possible verify the correct
homology arms by sequencing)
2) The Red/ET reaction did not take place because
o no or the wrong type of arabinose was used for induction (please make
sure you use L-arabinose!)
o in very rare cases increasing the time for recombination from 70 min
(incubation after electroporation) to up to four hours is necessary for
successful recombination.
11 BioVector NTCC Inc. – Recombineering-proficient E.coli Strain GB08-red, Version 1.0 (October 2012)
3) Problems with and after the electroporation:
o cells are not competent enough to take up the linear DNA. Please make
sure that the cells were kept on ice and that the water (or 10% glycerol)
is sufficiently cold. Linear DNA has been shown to be about 104
-fold
less active than DNA transformed in circular form (Eppendorf Operation
Manual Electroporator 2510 version 1.0). Make sure that the time
constant is around 5 ms.
o make sure that there is no arching during the electroporation process.
o make sure that after electroporation the cells are plated on the
appropriate concentration of antibiotics depending on the copy number
of the plasmid or BAC.
Similar number of colonies on both plates, the induced and the uninduced one:
A similar number of colonies on both plates (induced and uninduced one) indicates
that there are still traces of the circular (or supercoiled) plasmid used for preparing
the linear plasmid backbone left in the sample. Since the transformation efficiency of
linear fragments is 104
-fold less than that of circular DNA molecules you may obtain a
background even if no traces were visible on an agarose gel.
If the linear DNA fragment was obtained by restriction digestion, use less DNA and
gel purify the fragment! If the linear fragment was obtained by PCR, set up a DpnI
digest for your PCR product and gel purify it at the end!
You cannot separate the recombined plasmid from the non-recombined one after
recombination even after re-transformation (high copy plasmid!):
In very rare cases we have observed that after recombination it is difficult to separate
the original plasmid from the recombined one. If you cannot separate them by dilution
of the plasmid and re-transformation, you can choose a single cutting restriction
enzyme and digest the plasmid for a few minutes. After re-transformation the two
plasmids should be separated even when they were tangled before
12
6 References and Patents
6.1 References
• Zhang, Y., Buchholz, F., Muyrers, J.P.P., and Stewart, A.F. A new logic for
DNA engineering using recombination in Escherichia coli. Nature Genetics 20,
123-128 (1998).
• Angrand P.O., Daigle N., van der Hoeven, F., Scholer, H.R. and Stewart, A.F.
Simplified generation of targeting constructs using ET recombination. Nucleic
Acids Res 27, e16 (1999).
• Muyrers, J.P.P., Zhang, Y., Testa, G. and Stewart, A.F. Rapid modification of
bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27,
1555-1557 (1999).
• Muyrers, J.P.P., Zhang, Y., Buchholz, F. and Stewart, A.F. RecE/RecT and
Redα/Redβ initiate double-stranded break repair by specifically interacting
with their respective partners. Genes Dev 14, 1971-1982 (2000).
• Muyrers, J.P.P., Zhang, Y., Benes V., Testa, G., Ansorge, W. and Stewart,
A.F. Point mutation of bacterial artificial chromosomes by ET recombination.
EMBO Reports 1, 239-243 (2000).
• Zhang, Y., Muyrers, J.P.P., Testa, G. and Stewart, A.F. DNA cloning by
homologous recombination in Escherichia coli. Nature Biotech. 18, 1314-1317
(2000).
• Muyrers, J.P.P., Zhang, Y. and Stewart, A.F. Recombinogenic engineering:
new options for cloning and manipulating DNA. Trends in Bioch. Sci. 26, 325-
31 (2001).
• Fu,J., Teucher, M., Anastassiadis, K., Skarnes, W. and Stewart A.F. A
recombineering pipeline to make conditional targeting constructs. Methods in
Enzymology 477, 125-144 (2010).
• Fu, J., Bian, X., Hu, S., Wang, H., Huang, F., Seibert, P.M., Plaza, A., Xia, L.,
Müller, R., Stewart, A.F. and Zhang, Y. Full-length RecE enhances linear linear homologous recombination and facilitates direct cloning for
bioprospecting. Nature biotechnology. 30, 440-448 (2012).
13 BioVector NTCC Inc. – Recombineering-proficient E.coli Strain GB08-red, Version 1.0 (October 2012)
6.2 Patents
Red/ET recombination is covered by one or several of the following patents,
patent applications and related ones:
• Stewart, A.F., Zhang, Y., and Buchholz, F. 1998. Novel DNA cloning method
relying on the E. coli RecE/RecT recombination system. European Patent
No.1034260; United States Patent No 6509156.
• Stewart, A.F., Zhang, Y., and Muyrers, J.P.P. 1999. Methods and
compositions for directed cloning and subcloning using homologous
recombination. European Patent No.1204740; United States Patent No.
6355412.
• Zhang, Y., Fu, J., and Stewart, A.F. 2011. Direct Cloning. WO2011/154927

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