Protocol: Precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli
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Plant genome sequence data now provide opportunities to conduct molecular genetic studies at the level of the whole gene locus and above. Such studies will be greatly facilitated by adopting and developing further the new generation of genetic engineering tools, based on homologous recombination cloning in Escherichia coli, which are free from the constraints imposed by the availability of suitably positioned restriction sites. Here we describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements.
KeywordsBacterial Artificial Chromosome Luria Bertani Bacterial Artificial Chromosome Clone Locus Rescue Phaseolus Coccineus
BAC = Bacterial artificial chromosome
bp = base pairs
FIGE = field inversion gel electrophoresis
- GA 2ox
GA 2ox = gibberellin 2-oxidase
GFP = gree fluorescent protein
GOI = gene of interest
HA = homology arm(s)
HR = homologous recombination
LB = Luria Bertani medium
LR = Locus rescue
mPGK = mouse phosphoglycerate kinase promoter
OD = optical density
PCR = polymerase chain reaction
ORF = open reading frame
- rpm = revolutions per minute
UV = ultraviolet.
Plant bacterial artificial chromosome (BAC) resources are being generated for ever increasing numbers of species, providing scientists with long-range physical maps and associated sequence data for both model and crop plants. This provides opportunities for reverse genetics and functional studies at the level of the gene locus and above. The latter requires methods for the cloning and manipulation of large DNA fragments, without the limitations imposed by the need for suitably positioned restriction enzyme sites. Significant advances in this respect arose from the development of homologous recombination (HR) cloning in Escherichia coli, based on RecE/RecT (ET) [1, 2] and λ RED operon gene products [3, 4]. Essentially, in ET-based strategies, PCR-amplified linear DNA fragments with short regions of homology (~50 bp to 60 bp) are precisely targeted into any DNA sequence including high copy number plasmids, the E. coli chromosome and BACs. RED-based protocols rely on a defective λ prophage to provide functions that protect and recombine the linear DNA fragments, under the control of a temperature sensitive λ cl-repressor, with recombinogenic functions switched on at 42°C and off at 32°C. This fixed induction window helps to reduce unwanted rearrangements, allowing DNA to be stably cloned.
Our interest in long-range HR cloning was driven by a desire to create plant-specific tools and transgene constructs that target expression to the shoot apical meristem. We wanted to express the bean (Phaseolus coccineus) GAPc2ox1 (encoding GA 2-OXIDASE 1, which degrades bioactive gibberellin) in the shoot apex of sugar beet (Beta vulgaris) plants and study the effect on flowering. We present details of our constructs and the molecular tools (plasmids) developed to create these constructs by RED cloning.
• E. coli strain EL250 (genotype DH10B [λcI857(cro-bioA)<> ara C-PBADflpe] where <> indicates that cro-bioA has been substituted with ara C-PBADflpe) available from the authors of  who have developed a number of different strains including EL350 (with inducible araC-PBADcre). These strains carry a defective λ prophage with red and gam recombination genes under the control of the λPL promoter and exo and bet tightly controlled by the temperature sensitive cI857 repressor. Exo and Beta provide recombinogenic function while Gam inhibits the E. coli RecBCD nuclease from degrading electroprated linear DNA fragments. The promoter of the ara BAD operon (PBAD) is induced by L-arabinose for flpe and cre expression enabling removal of sequences between FRT and Lox P sites respectively. We used EL250 to enable removal of the kanamycin gene in our FRT-mPGK-Tn5-neo-FRT cassette. OUR RESULTS: The marker gene was removed as described  and worked with 90%–100% efficiency and we were able to recover 100 s of colonies which had become kanamycin sensitive.
• Luria Bertani (LB) broth and plates supplemented with antibiotics as required
• Fully sequenced BAC, PAC or other clones with desired gene locus. Plant BAC and PAC clones are widely available from a number of different sources, including individual labs and organisations e.g. The Arabidopsis Biological Resource Centre (ABRC) http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm or the Nottingham Arabidopsis Stock Centre (NASC) at http://arabidopsis.info/ and, http://www.dna.affrc.go.jp/ for rice genes and others. The AtSTM locus used in our experiments is cloned in BAC F24o1, sourced from the Arabidopsis Biological Resource Centre, Columbus, Ohio.
• pUC-based vectors to be used for making (i) the locus rescue gap-repair construct (must be counter selectable to the BAC/PAC), and (ii) the gene of interest (GOI) targeting cassette construct (must contain a counter selectable marker to the gap-repair construct).
• High fidelity Taq DNA polymerase. Preferably one which retains A-tails for TA cloning, e.g. the Expand High Fidelity PCR system (Roche Diagnostics).
• PCR primers – four locus-specific primers to amplify DNA fragments at the locus border flanks for the gap-repair rescue construct and two target site-specific primers (minimum 70 bp long) to generate GOI targeting products with destination site specific 5' and 3' homology arms.
• PCR product and gel purification kits e.g. the Qiagen QIAquick™ range and Dpn I restriction enzyme – used to remove plasmid templates from PCR reactions because it only cleaves methylated sites.
• General reagents for standard gene cloning and gel electrophoresis
• Orbital shaking incubator
• Orbital shaking water bath e.g. Grant OLS 200 – essential for induction of recombination functions in bacterial cells.
• Electroporator e.g. Bio-Rad E. coli Pulser
• PCR Machine
• Long wave UV transilluminator – long wave ultra violet light is less damaging to DNA during excision of bands from gels. UV-damaged DNA will not recombine efficiently.
• Electrophoresis equipment capable of field inversion gel electrophoresis (FIGE) or pulsed field gel electrophoresis (PFGE) e.g. BioRad CHEF DR-II, DR-III or Mapper™ XA, for efficient resolution of large DNA fragments
• Spectrophotometer for cell density quantification
• Temperature controlled centrifuge able to run at 4°C
The protocols outlined below describe the development of (i) an AtSTM-locus specific gap-repair rescue vector, (ii) a plant gene targeting construct with a removable kanamycin resistance marker cassette from pGK-FRT , under the control of both the bacterial Tn5 promoter and the mouse phosphoglycerate kinase (mPGK) promoter for selection in prokaryotes and eukaryotes respectively. This provides templates for PCR amplification of selectable gene fragments that can be precisely targeted into any desired gene locus; and (iii) a bean (Phaseolus coccineus) GAPc2ox1 transformation construct, co-ordinately driven by "all" AtSTM locus elements, designated pSTM 17::GAPc2ox1. We have also constructed a pENTR4-based AtSTM gap-repair rescue vector for the production of a Gateway™ (Invitrogen) compatible entry clone and generic T-DNA transformation constructs as well as an mgfp5-ER targeting cassette. The pSTM 17::GAPc2ox1 was successfully transformed into sugar beet, demonstrating for the first time that the mouse PGK promoter is fully functional in transgenic plants, thus enabling the direct exploitation of existing mammalian tools.
Key steps in the EL250 RED-HR locus rescue and engineering procedure
1. Design of PCR primers for amplification of locus rescue (retrieval) homology arms and also for GOI targeting
2. Construction of a gap-repair locus rescue vector.
3. Construction of a targeting vector containing the GOI upstream of a counter selectable marker (different from that in the gap-repair construct).
4. Electroporation of EL250 cells with the BAC or clone containing the desired gene locus and preparation of electrocompetent BAC/EL250 cells induced for Exo, Beta and Gam functions.
5. Performance of gap-repair locus rescue, in cells treated as above; selection of recombinants and confirmation by restriction digestion analysis and sequencing. Transformation of the rescued locus plasmid into fresh EL250 cells.
6. PCR amplification, purification and quantification of the GOI targeting cassette and its site-specific recombination into the rescued locus plasmid in EL250 cells. Selection and confirmation of recombinants as above.
The recombineered plasmid is now ready for application in functional analyses as desired.
Primer design and plasmid constructs
For the GOI targeting cassette, primers must include at least 30 to 50 bp at the 5' end, to provide homology arms for site-specific recombination. The target site sequence must not have any mismatches as this will inhibit recombination. It is therefore essential to source primers from suppliers able to guarantee sequences of long primers. Our primers were custom made by Sigma Genosys.
IN OUR HANDS: Creation of these basic plasmids was the key limiting step as it is dependent on conventional cloning and therefore, on the availability of suitably placed restriction sites. However, once constructed, the gene targeting constructs can be used to target the expression cassette into any desired site whereas the gap-repair construct is suitable only for subcloning the specific gene fragment/locus. Our targeting construct backbone has therefore been designed to include a plant-specific polyA signal (Nopaline synthase (nos) termination sequence), for generic use with any plant cDNA sequence.
AtSTM gap-repair construct
Using BAC F24o1 DNA template (represented in Fig. 2A), and the Expand High Fidelity DNA polymerase PCR system (Roche Diagnostics) we amplified 564 bp (incorporating 5'Sal I and 3'Sph I sites) and 479 bp (incorporating 5'Sph I and 3'Hind III sites) homology arms respectively at the downstream and upstream flanks of the AtSTM locus (Fig. 2B). At the start of our project, the pentatricopeptide repeat protein (PPR) downstream of the STM coding sequence was annotated as a predicted ORF and we therefore opted to include it in the STM locus fragment. Now, it would be excluded as the locus boundary. PCR reactions included primers Hind III 3' hyp/Sph I 5' hyp or Sprot H1 Sal I/Sprot H1 Sph I at 0.3 μM each and were incubated for 1 cycle at 94°C for 2 min. followed by 30 cycles of 94°C for 15 sec; 64°C for 30 sec; 72°C for 1.5 min; and 1 cycle of 72°C for 5 min. The individual products were then sub-cloned into pGEM-T Easy (Promega), recovered and cloned into pBluescript II SK+ (Stratagene) in a three-way ligation reaction, to create the gap-repair construct.
NOTE : In our hands, cloning of PCR products is more efficient if we shuttle them via a PCR cloning vector. Any TA cloning vector is suitable. In this case, it is important to ensure that the proof reading activity of the Taq Polymerase used does not remove A-tails.
Targeting construct backbone
GAPc2ox targeting construct
The full length GAPc2ox1 cDNA in plasmid pST33 (a kind gift from Drs Andy Philips and Peter Hedden, Rothamsted Research) was excised with Spe I/Xho I and cloned into the Nhe I/Sal I site of pNosTerFRT-neo (Fig. 3Bi).
mgfp5-ER targeting cassette
The mgfp5-ER gene (GenBank U87974) was isolated from pBIN35S-mgfp5-ER (a kind gift from Dr Jim Haseloff, Department of Plant Sciences, University of Cambridge, UK) as a Bam HI/Sac I fragment and cloned into the Sma I site of pNosTerFRT-neo (Fig. 3Bii)
WARNING! GUS reporter cassettes are not suitable as they are able to recombine with the endogenous (chromosomal) E. coli gene during HR cloning.
RED Cloning Protocol
Original methods and information on how to obtain host cells can be found at the recombineering website http://recombineering.ncifcrf.gov/
A: Preparation of electrocompetent EL250 cells
These cells will be periodically used to receive new plasmids as they are constructed and required for recombineering. It is therefore advisable to make and store a sizable batch.
1. Streak out cells on LB plates and grow at 32°C. The cells are temperature sensitive and will die at 37°C
2. Inoculate a single colony into 5 ml LB and grow overnight.
3. Inoculate 1 ml of the overnight culture into 50 ml LB in a 500 ml flask and grow at 32°C with shaking at 200 revolutions per minute (rpm) until the cells density has reached OD600 = 0.5 – 0.8.
4. Spin cells at 4°C (rotor must be pre-cooled) and wash with 5 ml ice cold sterile distilled water. Spin and discard supernatant. Repeat wash with 5 ml aliquots of ice cold SDW two more times.
5. Finally resuspend cells in 500 μl of ice cold sterile distilled water and aliquot 100 μl lots into cooled 1.5 ml microfuge tubes.
NOTE : Cells can be used immediately or re-suspended in ice cold sterile 10% (v/v) glycerol and stored at -80°C until required.
B: Transformation of BAC F24o1 and induction of recombinogenic function in EL250
1. On ice, add 10 ng-100 ng of F24o1 DNA to 50 μl of competent EL250 cells. Mix by gentle pipetting and transfer to a pre-cooled 0.1 cm electroporation cuvette.
2. Pulse at 1.75 kV in a Bio-Rad E. coli Gene Pulser. Immediately add 1 ml LB broth and incubate at 32°C for 1–1.5 h in a shaking incubator set at 200 rpm
3. Plate cells on LB kanamycin and select for transformants. NOTE: It is advisable at this stage to check the integrity of the BAC clone by restriction digestion analysis, to ensure that there have been no rearrangements.
4. Grow F24o1/EL250 cells as described in A: steps 1 – 3 except that all LB media must be supplemented with kanamycin (or relevant antibiotic) to select for the BAC. NOTE: before the next step, ensure that the shaking water bath is switched on early and stabilised at 42°C ready for use and pre-warm the conical flask. An ice slurry bath must also be made ready – DO NOT USE JUST ICE – it will not cool cells fast enough.
5. For induction, transfer 10 ml of the growing culture into a pre-warmed 250 ml conical flask and incubate in the water bath at 42 °C with shaking at 200 rpm for a total of 15 min. NOTE : Keep the remaining 40 ml of culture at 32 °C to act as a non-induced control.
6. Immediately after 15 min. place the flask in the ice slurry bath and swirl by hand to quickly cool down the cells. Include a similar flask with 10 ml of non-induced control cells – this will be cooled down and treated in the same way as the test cells from now on. NOTE: (i) Induced cells must be used immediately as they will lose activity above 0°C. Therefore it is important to work quickly from now on. However, cells may be kept on ice for a total of 40 min. without significant loss of activity. (ii) Ensure that the centrifuge and rotor are pre-cooled to 4 °C before the next step.
7. Centrifuge the 10 ml aliquots of induced and control cells for 8 min at 5500 g and at 4 °C. Retrieve pellets and wash three times in 1 ml ice cold sterile distilled water and centrifuge as above. NOTE: To save time, washing steps can be carried out in 1.5 ml microfuge tubes keeping everything ice cold and centrifuging at 4 °C for 20 seconds each time.
8. After final wash, re-suspend the cell pellet in 100 μl of ice cold sterile distilled water. This is enough for two electroporation transformation reactions.
C: AtSTM locus rescue from BAC F24o1 by gap-repair HR
Before starting: Ensure that purified and linearised gap-repair vector is available at concentrations suitable to deliver 10 to 100 ng in volumes up to 10 μl. We strongly recommend gel quantification with known standards as we find this more accurate than OD260 nm measurements.
N.B. All HR experiments should be carried out with the induced and un-induced control cells in parallel.
1. Using linearised gap-repair construct DNA, electroporate induced competent F24o1/EL250 cells as described in B: steps 1 – 2.
2. Select recombinants on LB supplemented with antibiotic marker for the gap-repair vector. We used pBluescriptII KS+ and therefore selected on LB ampicillin. The use of pBluescript also limits the size of insert which can be rescued and 17 kb was the largest fragment we were able to retrieve by gap repair HR cloning.
TROUBLE SHOOTING: Growth of un-induced colonies on selective plates suggests incomplete digestion of the gap-repair construct during linearization. However, the number of colonies should be low (we typically recovered 5 – 10 colonies from un-induced cells). Otherwise repeat with improved digestion and/or gel purification of the linearised gap repair construct.
4. Transform the rescued plasmid into fresh EL250 cells and prepare induced competent cells as described, ready for the locus targeting experiment. We designated these cells STM17/EL250 because they contained rescued ~17.5 kb of the AtSTM locus. NOTE: For our application, we use a protoplast based direct transformation method and therefore opted to use pBluescript as the backbone for our gap repair and eventual transformation construct. However, for Agrobacterium-based systems, we recommend using a Gateway compatible Entry vector (available from Invtrogen: ) as this will enable subsequent transfer of the captured, manipulated locus into a T-DNA binary destination vector for example the ones available from Plant Systems Biology http://www.invitrogen.com(VIB-Gent University: http://www.psb.rug.ac.be/gateway) or the pEarlyGates vectors, details of which can be found at the website: http://www.biology.wustl.edu/pikaard/pEarleyGate%20plasmid%20vectors/Table%20of%20vectors.html.
Recently, we have successfully created an AtSTM locus rescue vector based on the Invitrogen pENTR4 Gateway™ compatible vector in which we plan to capture/manipulate the locus as described and determine the success rate of transfer into a promoterless T-DNA destination vector pB7WG2Δ35S (based on pB7WG2 from VIB-Gent University). These are newly available resources that should enable the creation of constructs for the more generic Agrobacterium-mediated plant transformation systems.
WARNING!: Direct use of T-DNA vectors as gap-repair constructs in RED cloning although attractive, may prove problematic because of the common use of a limited number of identical or very similar promoter and polyA signal sequences, which if also present in the targeting cassette will result in illegitimate recombination events. For this reason, we did not attempt any experiments with T-DNA vectors, opting instead to go via the Gateway™ system as detailed above.
D: Replacement of AtSTM exon1 by in-frame fusion of the promotorless GAPc2ox-FRT-neo-FRT targeting cassette
Before starting: The purified, Dpn I treated and quantified PCR amplified GOI targeting cassette should be made ready for this experiment.
1. Using up to 100 ng of the PCR amplified GAPc2ox1-FRT-neo-FRT targeting cassette, electroporate induced STM17/EL250 cells as described in B: steps 1 – 2. The targeting cassette was amplified with HotStar Taq DNA polymerase (Qiagen) and primers 28001 frtlow and 2oxexon1 (0.3 μM each) in a 50 μl reaction volume. Incubation conditions were 1 cycle 95°C for 1 min followed by 20 cycles of 94°C for 15 sec; 68°C for 4.5 min. (with a 5 sec. time increment in each cycle); followed by 1 cycle of 68°C for 10 min.
TROUBLE SHOOTING: High un-induced colony numbers on selective plates suggest targeting cassette template contamination instead of recombination. Check Dpn I digests and use this in combination with gel purification to remove template DNA from the target cassette PCR product prior to electoporating for HR. In our experience, if the number of colonies from un-induced cells is at least 50 – 100 fold less than from the induced cells, then it was worth screening colonies from induced cells.
Manipulation of large DNA fragments to make complex constructs for functional genomics or genetic engineering for crop improvement is possible using HR cloning in E. coli. We have successfully used HR cloning in E. coli to sub-clone the Arabidopsis thaliana SHOOTMERISTEMLESS (STM) gene locus from a BAC clone into pBluescript and to replace exon 1 sequences with a Gibberellin 2-oxidase cDNA gene-of-interest cassette tightly linked to an FRT-flanked kanamycin selection marker gene. This cassette is of generic use because firstly, it can be targeted/recombineered into any locus/destination site. Secondly, the kanamycin resistance gene is under the control of both the bacterial Tn5 promoter and the mouse phosphoglycerate kinase promoter (mPGK), which respectively allow for selection in prokaryotes and eukaryotes. We have now demonstrated the utility of the mPGK promoter for driving expression in transgenic plants and this suggests that there may well be increased scope for plant scientists to directly benefit from existing molecular genetic tools developed for application in the biomedical field.
E. coli ET- and RED-HR cloning are well established technologies within the biomedical field and they have many uses besides the creation of transformation constructs with long-range regulatory elements. The identification of regulatory elements or locus control regions located at a distance from the gene sequence can be assisted by this strategy. Point mutations, deletions or insertions, gene fusions and antisense constructs can be engineered on any BAC for functional genomics studies. The scope for plant science is further enhanced by the recently reported application of HR to convert BACs into binary vectors  together with (i) the availability of a BAC-based physical map of A. thaliana, (ii) freely available genome sequence information through the Arabidopsis Genome Initiative, (iii) access to rice sequence data and BAC resources through The Institute for Genomic Research (TIGR) and the Rice Genome Resource Center (RGP).
Resources for RED/ET cloning are available from Neal Copeland and Nancy Jenkins for both profit and non-profit organisations. Details can be found at the following website: http://recombineering.ncifcrf.gov/reagent_request.asp. The commercial company GeneBridges http://www.genebridges.com/web/company/index.htm also offers reagents and a DNA engineering service.
Ann Mathews, Roz Williamson and Sarah Yallop for technical assistance molecular analyses and sugar beet transformation.
The project was funded by the Biotechnology and Biological Sciences Research Council of the UK as part of the ROPA scheme.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.