Mutagenesis of cloned moss genes using mini-transposon constructs
(Construction of Tagged mutant library by shuttle mutagenesis)

Tomoaki Nishiyama, Yuji Hiwatashi, and Mitsuyasu Hasebe

National Institute for Basic Biology
38 Nishigonaka, Myodaiji-cho
Okazaki 444-8585
Japan
Tel: +81-564-55-7546
FAX: +81-564-55-7546
e-mail: mhasebe@nibb.ac.jp

Outline

Shuttle mutagenesis is aimed to make a tagged mutant library for the large-scale characterization of gene in the moss Physcomitrella patens. This method involves essentially the following three steps.

  1. generation of Physcomitrella genomic library in E. coli.
  2. insertion of mini-transposons in the moss genomic library in E. coli.
  3. transformation of the moss with the mutagenized (tagged with the mini-transposon) genomic library.

The transformation is expected to result from recombination between the moss DNA sequences flanking the transposon and homologous genomic sequences. The genomic sequences should be replaced by the tagged homologous sequences, and tagged mutants of P. patens are obtained.

This system is also useful for the disruption of any specific gene via homologous recombination. In this case, a single genomic clone may be used instead of the genomic library.

This protocol is modifed from the method established for yeast system by Seifert et al. (1986), Burns et al. (1994), and Ross-Macdonald et al. (1997). We appreciate Petra Ross-Macdonald (Yale Univ.), Michael Snyder (Yale Univ.), Akio Tohe (Univ. Tokyo), and Kenjiro Fujiwara (Univ. Tokyo) for their kind supply of materials used in yeast system and valuable technical suggestions. The protocol of Physcomitrella transformation was kindly provided by David Cove (Univ. Leeds).

(1) Generation of Physcomitrella Genomic Library in E. coli

The moss genomic library is constructed in a plasmid vector suitable for the shuttle mutagenesis in E. coli. The size of inserted fragments should be between 3.5 kb and 6 kb, which is appropriate for efficient transposition and for homologous recombination of the moss at later step.

  1. Extract total DNA of Physcomitrella patens from protonema cells with CTAB method (Murray and Thompson 1980). Remove RNA with PEG precipitation after RNase treatment.

  2. Partial digestion with Sau3A1. The condition of the partial digestion should be determined by preliminary experiments [Sambrook et al. 1989, pp. 9.24-9.26].

  3. Size fractionation with agarose gel extraction. Run the digested DNA on 0.6% agarose gel with 1x TAE buffer, and slice out the gel block containing DNA fragments ranging between 3.5 kb and 6 kb in size. Extract the DNA fragments by electro-elution method [Sambrook et al. 1989, pp. 6.28-29, 34-35].

  4. Modify the terminal nucleotide sequences of the extracted DNA fragments to insert in the Sal1 site of the cloning vector (pHSS-Sal). The 5'-GATC-3' protruding ends of the extracted Sau3A frangments should be filled with dG and dA using Klenow Fragment (use one unit of enzyme per ug of DNA)[Sambrook et al. 1989, p. 9.29]. Purify with phenol extraction, chloroform extraction and ethanol precipitation.

  5. Preparation of vector.

    The pHSS-Sal plasmid contains a pBR322 replication origin, a Kanr gene, and a SalI cloning site between two NotI sites. The pHSS-Sal does not have the 38bp terminal sequence of Tn3, which is held in most vectors derived from pBR322. Closed circular form of pHSS-Sal is purified with CsCl density gradient centrifugation after alkali lysis. pHSS-Sal is completely digested with SalI, run on agarose gel, and recovered by electro elusion. The 5'-TCGA-3' protruding ends of the Sal1 digested pHSS-Sal vector should be filled with dT and dC using Klenow Fragment, and then dephosphorylated (we usually use Thermosensitive Bacterial Alkaline Phosphatase of Gibco/BRL). Purify with phenol extraction, chloroform extraction, and ethanol precipitation.

  6. Ligate the partially filled vector and genomic fragments using Takara ligation kit ver. 2. The vector to insert ration should be around 1:1, although it is better to confirm the ratio by pilot experiments.

  7. Transformation of E. coli.

    The moss genomic DNA is supposed to be methylated, and we may not be able to clone the moss genomic DNA using the E. coli with mcr+ and mrr+ genes. So, at the first step, we use the E. coli, XL2-blue MRF' (Stratagene, [Delta(mcrA) Delta(mcrCB-hsdSMR-mrr)]) lacking the genes (mcr and mrr). In addition to mrr and mcr genes, the XL2-blue MRF' strain lacks both restriction and modification genes (hsdR, hsdM: r-m-). Plasmids of the m- E. coli with the recognition sequence of the EcoK system are restricted in the hsdR+ (r+) E. coli, although we have to use E. coli NG135 (or NS2114Sm) strain (hsdR+: r+) for transposition in later experiments. So, we transferred the plasmids obtained from the XL2-blue MRF' (hsdR, hsdM: r-m-) to the DH5alpha (hsdR, hsdM+: r-m+). Then, the plasmids obtained from DH5alpha were used to transform the RDP146/pLB101 strain (hsdR+: r+).

    Prepare competent cells with the modified Hanahan method (Inoue et al. 1990). Electroporation may be better for high transformation efficiency of longer fragments.

    First, transform XL2-blue MRF' with the ligation product. Select the transformants on the LB plate containing Kan40. The transformants are grown on the plate for 16 hours at 37C. Gather all of transformed colonies on a plate, suspend in the Solution I (50mM Glucose, 25mM Tris-HCl [pH8.0], 10mM EDTA [pH8.0]) for the alkali lysis method, and extract plasmids. Several hundreds of colonies are usually collected in each experiment. The extracted plasmids sometimes contain no-inserted plasmids showing the same size as the intact pHSS-Sal on agarose electrophoresis. The size of the vector pHSS-Sal in itself is 2.2kb. On 0.6% agarose gel, closed circular form of the pHSS-Sal comes around 1.6 kb, and the open circular form comes between 2kb and 3kb. An additional broad band was visible between 3.3 kb and 4.5 kb, which presumably corresponds to the closed circular form of the vector plus insert. The bands between 3kb and 7kb were cut and plasmids were recovered with GeneCleanIII kit. The ratio of inserted plasmids to no-inserted plasmids depends on each batch of dephosphorylated pHSS-Sal. Usually, the ratio is 1 to 1 according to the brightness of Ethidium bromide staining.

    Secondly, transform the DH5alpha strain with the inserted plasmids. Plasmids are further purified from the transformed DH5alpha using the same procedure as mentioned above. Plasmids obtained from the DH5alpha usually include much smaller amount of no-inserted plasmids than those from the first step, and it is not necessary to purify no-inserted plasmids by the agarose gel extraction. Use the extracted plasmids for the following transformation of RDP146/pLB101 without agarose gel extraction.

    Finally, transform the RDP146/pLB101 cells with the extracted plasmids from DH5alpha cells by electroporation (We have not been able to prepare competent cells of this strain with high competence by the modified Hanahan method.). The RDP146/pLB101 cell contains pLB101 plasmid containing Camr gene and transposase gene (tnpA), and the transformants should be selected on LB plate with Kan40 and Cam34.

(2) insertion of modified mini-transposons in the moss genomic library in E. coli (Seifert et al. 1986)

Hereafter the procedure follows the protocol by Yale Genome Analysis Center (YGAC), which is available at http://ycmi.med.yale.edu/YGAC/protocol.html.

Five kinds of transposons are available depending on purposes (see Appendix). All transposons contain Ampr gene and located on the F derivative pOX38. E. coli with the F episome containing a transposon is selected on Amp50 plate.

This protocol is applicable for the derived mini-transposon with the res site, which is the target sequence of resolvase. Transposons in Appendix except mTn-nptII are this type. The mTn-nptII does not have the res site and Cre recombinase should be used to resolve at the loxP site. For the mTn-nptII transposon, use E. coli NS2114sm cell instead of NG135 cell.

  1. Mating of the transformant E. coli (RDP146/pLB101) to E. coli carrying the transposon on the F derivative (RDP146/pOX38::mTn-*).

    1. Preparation of E. coli with the transposon(RDP146/pOX38::mTn-*)

      E. coli with a transposon (RDP146/pOX38::mTn-*) should be streaked on LB plate containing Amp50 the day before transforming RDP146/pLB101 with the library. On the day of transformation, inoculate a single colony in 2-5 ml of LB medium (with or without ampicillin). Next morning, subculture the RDP146/pOX38::mTn-* of 1:100 dilutions in fresh liquid LB without antibiotics.

    2. Preparation of library strain

      Grow the transformed RDP146/pLB101 overnight on the plate. Elute colonies from the plate: put 3 ml of LB medium on the plate, scrape off the colonies with a spreader. Dilute the eluted cells to the same density as the subcultured RDP146/pOX38::mTn-*. Grow at 37C to early log phase (about 2 hours).

    3. Mating of the transposon strain (RDP146/pOX38::mTn-*) and the library strain.

      Both strains should be grown for 2 hours at 37C. For mating, the recipient (the library strain) may be denser, but the donor (RDP146/pOX38::mTn-*) should not be overgrown. Mix 600ul of the library strain and 300ul of the RDP146/pOX38::mTn-* strain, and incubate at 37C without agitation for 20min. The F derivative moves from the RDP146/pOX38::mTn-* cells to the library strain (RDP146/pLB101/pHSS-Sal-moss DNA). The bacteria with the F derivative (Ampr), pLB101 (Cmr), and pHSS-Sal-moss DNA (Kanr) are selected by three antibiotics: Amp50 Kan40 Cm34. Plate as 100 ul aliquots of original, 10-fold dilution, and 100-fold dilution onto LB plate with Amp50 Kan40 Cm34. Usually 10-fold dilution gives a good density of transconjugate (the cell that received F episome via conjugation). To confirm the experiments, spot the starting strains (the transposon strain (pOX38::mTn-*) and the library strain (RDP146/pLB101/pHSS6-Sal-moss DNA) on LB Amp50 Kan40 Cm34 plate. These strains should not grow on the plate.

  2. Transposition of mTn-* to pHSS-Sal-moss DNA

    Incubate the plate with transconjugates for 1-2 days at 30C. During the culture at 30C, the transposon is integrated in the pHSS-Sal-moss DNA plasmid.

  3. Resolution of the co-integrate.

    1. Preparation of NG135/pNG54

      NG135/pNG54 is resistant to streptomycin and has the plasmid pNG54 (pACYC184 with mTn3 res and tnpR seqs; active resolvase, chloramphenicol resistant). Streak the freeze stock of NG135/pNG54 on LB plate containing Sm100. Inoculate a single colony in liquid LB media (with or without antibiotics). Shake at 37C overnight. Next morning, subculture the bacteria 1:100 dilutions in fresh liquid LB without antibiotics. Culture until early log phase.

    2. Preparation of RDP146/pLB101 containing the cointegrated F derivative with pHSS-Sal-moss DNA plasmid.

      Elute colonies from plates: put 2 ml of LB on the plate, scrape off the colonies with a spreader. At least, several thousands colonies should grow on a plate. Dilute the eluted cells to roughly the same density as the subcultured NG135/pNG54.

    3. Mating and resolving

      When the donor (RDP146/pLB101 containing the cointegrated F derivative with pHSS-Sal-moss DNA plasmid) reached early log phase, mix 600ul of the recipient (NG135) and 300ul of the donor, and incubate at 37C without agitation for 20min. The cointegrated F derivatives with pHSS-Sal-moss DNA move from the donor cells to the recipient cells. Plate 100ul aliquots on LB Amp50 Kan40 Sm100, and grow overnight at 37C. During this culture, the cointegrated F derivatives with pHSS-Sal-moss DNA plasmid are resolved by the resolvase. To confirm the experiments, spot each original strain before mating (NS2114Sm and the RDP146/pLB101 containing the cointegrated F derivative with pHSS-Sal-moss DNA plasmid) on LB Amp50 Kan40 Sm100 plate. These strains should not grow on the plate.

      After resolving, the NG135 cells contain both the original F derivative (pOX38::mTn-*) and the transposed plasmid (pHSS-Sal-moss DNA with a transposon insertion). One copy of the original F derivative is present in a single NG135 cell, while multiple transposed plasmids are present in a single NG135 cell, because the pHSS-Sal plasmid contains pBR322 replication origin.

    4. Extraction of the resolved plasmid from the NG135

      Elute colonies from plates: put 2 ml of LB on the plate, scrape off the colonies with a spreader. At least, several thousands colonies should grow on a plate. Extract the plasmids with alkaline lysis method.

  4. Transform usual lab strain with the transposed pHSS-Sal-moss DNA.

    As the quality of extracted DNA from NG135 is usually bad, usual lab strain (e.g. XL2-blue MRF', DH5alpha) should be transformed with the recovered plasmids (the transposed pHSS-Sal-moss DNA plasmids). XL2-blue MRF' or DH5alpha give a good quality of DNA because of low nuclease activity. The transformed colonies are selected with Amp50, Kan40. After an overnight growth at 37C, elute colonies from the LB plates with 2 ml of LB, and scrape off the colonies with a spreader. The plasmids are purified with polyethylene glycol precipitation after alkali lysis.

(3) Transformation of the moss with the mutagenized (tagged with the transposon) genomic library.

  1. Completely digest the extracted plasmids with NotI to divide them into the vector and inserted DNA fragments. Purify with phenol extraction, chloroform extraction, and ethanol precipitation. Dissolve in TE (10mM Tris-HCl [pH8.0], 1mM EDTA [pH8.0]) and adjust the concentration to 1.0 ug/ul.

  2. Transform Physcomitrella protoplasts with the digested plasmids according to Schaefer et al. (1991).

    Transformation with 30ug of plasmid gives the largest number of stable transformants, although multiple bands are usually detected by genomic southern hybridization with the nptII gene as a probe. Experiments are in progress to see whether the multiple transposons are inserted in multiple sites of the moss genome, or multiple transposons are inserted in a single (or a few) site. Based on preliminary experiments, most of transposons are likely inserted in a few genome sites.

    Although reducing the amount of plasmid for the transformation will give less number of transformants, less insertions of transpoons per transformant as assessed by southern analysis. We are now usually using 3 to 6 ug of DNA per transformation.

    Our transformation protocol is a mixture of the Leads and Lausanne protocol.

Appendix:

1. antibiotics

Ampicillin
Amp, 50 mg/ ml in water. Use at 50 mg/l (Amp50)
Kanamycin
Kan, 10 mg/ ml in water. Use at 40 mg/l (Kan40)
Chloramphenicol
Cm, 34 mg/ml in ethanol. Use at 34 mg/l (Cm34)
Streptomycin
Sm, 10 mg/ml in water. Use at 100 mg/l (Sm100)

2. E. coli strains

* RDP146/pLB101, NS2114sm, pHSS6, and pTn are kindly provided by K. Fujiwara (originally from Seifert lab (Northwestern University)).

* NG135/pNG54, RDP146, and plasmids having minitransposon with HA are kindly provided by Dr. Michael Snyder (Yale Univ.)

3. vector

pHSS-Sal (modified from pHSS6 as described in Burns et al. 1994)

4. transposons

Following transposons are located on the F derivative, pOX38.

Plasmids containing the transposons are also available (pTn-nptII, pTn-4HA-nptII-Amp, pTn-3xHA-nptII-Amp, pTn3-diaGT1, ?mTn-3XHA-nptII-Amp). Original transposons were kindly provided by Seifert lab (pTn) and Dr. Michael Snyder (pTn-4xHA, pTn-3xHA; Yale Univ.).

4-1. pOX38::mTn-nptII

Uses: Gene disruption

mTn-nptII was constructed by modifying the pTn3 (Seifert 1986), and have similar structure as mTn-LEU2/lacZ (http://ycmi.med.yale.edu/YGAC/lacZ_LEU2_info_p.html). This transposon can easily be inserted at a random site in a DNA fragment cloned in pHSS-Sal or other equivalent plasmids (pHSS6, pHSS8). The mutagenized DNA is then transformed into the moss, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles.

4-2. pOX38::mTn-4HA-nptII-Amp

Uses: Gene disruption, analysis of gene expression, HAT epitope-tagging protein at range of sites.

mTn-4HA-nptII-Amp was constructed by modifying the mTn-4xHA/lacZ transposon (Ross-Macdonald et al. 1997: http://ycmi.med.yale.edu/YGAC/4xHA_lacZ_info_p.html). This transposon can easily be inserted at a random site in a DNA fragment cloned in pHSS-Sal or other equivalent plasmids (pHSS6, pHSS8). The mutagenized DNA is then transformed into the moss, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. When the transposon is fused in-frame to a gene, or inserted in an intron, expression of the gene is monitored with GUS staining, because Physcomitrella patens cdc2 intron (kindly supplied by K. Fujiwara, Univ. Tokyo) is fused to the 5' end of uidA gene, which encodes beta-glucronidase. The transposon can be excised by Cre mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus a 262-bp insertion containing sequences encoding 4 copies of the HA epitope.

4-3. pOX38::mTn-3xHA-nptII-Amp

Uses: Gene disruption, analysis of gene expression, HAT epitope-tagging protein at range of sites.

mTn-3xHA-nptII-Amp was constructed by modifying the mTn-3xHA/lacZ transposon (Ross-Macdonald et al. 1997: http://ycmi.med.yale.edu/YGAC/3xHA_lacZ_info_p.html). This transposon can easily be inserted at random site in a DNA fragment cloned in pHSS-Sal or other equivalent plasmids (pHSS6, pHSS8). The mutagenized DNA is then transformed into the moss, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. When the transposon is fused in-frame to a gene, or inserted in an intron, expression of the gene is monitored with GUS staining, because Physcomitrella patens cdc2 intron (kindly supplied by K. Fujiwara, Univ. Tokyo) is fused to the 5' end of uidA gene. The transposon can also be excised by Cre mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus a 274-bp insertion containing sequences encoding the 3xHA tag and the factor Xa protease cleavage recognition site.

4-4. pOX38::mTn3-diaGT1

Uses: Gene disruption, analysis of gene expression, HAT epitope-tagging protein at range of sites.

pTn3-diaGT1 was constructed by modifying the mTn-3xHA/lacZ transposon (Ross-Macdonald et al. 1997: http://ycmi.med.yale.edu/YGAC/3xHA_lacZ_info_p.html). This transposon can easily be inserted at a random site in a DNA fragment cloned in pHSS-Sal or other equivalent plasmids (pHSS6, pHSS8). The mutagenized DNA is then transformed into the moss, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. When the transposon is fused in-frame to a gene, or inserted in an intron, expression of the gene is monitored with GUS staining, because the 4th intron of Arabidopsis G protein (kindly supplied by Dr. R. Martienssen, Cold Spring Harbor Lab.; Sundaresan et al. 1995) is fused to the 5' end of uidA gene. The transposon can be excised by Cre-mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus insertion containing sequences encoding 3 copies of the HA epitope.

4-5. pOX38::mTn-3XHA-nptII-Amp

Uses: Gene disruption, analysis of promoter activity (enhancer trap)

mTn-3XHA-nptII-Amp was constructed by modifying the mTn-3xHA/lacZ transposon (Ross-Macdonald et al. 1997: http://ycmi.med.yale.edu/YGAC/3xHA_lacZ_info_p.html). This transposon can easily be inserted at a random site in a DNA fragment cloned in pHSS-Sal or other equivalent plasmids (pHSS6, pHSS8). The mutagenized DNA is then transformed into the moss, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. When the transposon is inserted around the enhancer sequences, activity of the enhancer is monitored with GUS staining, because the CMV35S minimal promoter is fused to the 5' end of uidA gene. The transposon can be excised by Cre-mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus insertion containing sequences encoding 3 copies of the HA epitope.

References


Tomoaki Nishiyama, Yuji Hiwatashi, and Mitsuyasu Hasebe
National Institute for Basic Biology
38 Nishigonaka, Myodaiji-cho
Okazaki 444-8585
Japan
Tel: +81-564-55-7546
FAX: +81-564-55-7546
e-mail: mhasebe@nibb.ac.jp