Triparental conjugation protocol for Nostoc punctiforme ATCC 29133                                                5/2000

Used for transposon mutagenesis or gene replacement in the chromosome

When transferring DNA into Nostoc for either recombination or transposition purposes, conjugation is the 
preferred method.  For transferring replicating plasmids into Nostoc, see the Electroporation protocol.  
Conjugation into Nostoc usually requires a triparental mating.  The three strains required are (i) an 
E. Coli strain carrying the mobilizer and helper plasmids (we typically use HB101 carrying both pRK2013
and pRL528), (ii) an E. coli strain carrying the donor plasmid (this can be pRL1063a which is the Tn5 
derivative used for mutagenesis, or derivatives of pRL271 carrying interrupted genes of interest for gene 
replacement in the chromosome by using the SacB system), (iii) and lastly Nostoc is required as the 
recipient.  See Cohen et al. 1994. Microbiology 140:3233-3240, or Cohen et al. 1998. Methods Enzymol. 
297:3-17 for a complete description of transposon mutagenesis protocol.  For gene replacement, see Cai 
and Wolk. 1990. J. Bacteriol. 172:3138-3145 for a description of the SacB system.

Protocol:

1.  Preparation of Nostoc
         a. Fragment an exponentially growing Nostoc culture by sonication to 4-6 cells/filament (we typically 
         use a microtip in a Heat System Sonicator at an output setting of 3 and 50% duty), pellet at 1,000 x g     
         (maximum rpm) in a clinical centrifuge for 10 min.
         
         b.  Resuspend the pellet in 50 ml of AA/4 medium containing 5 mM MOPS and 2.5 mM NH4Cl at pH = 7.8 
         and allow the cells to recover from sonication for several hours.

         c.  Just prior to conjugation, measure the density of the recovery culture by determining the Chlorophyll a 
         concentration
  
         d.  Concentrate the culture to obtain a density of 75-100 ug Chl a/ml (~ 0.75 - 1.0 x 108 cells/ml).

Note:  sonication is not necessary if clonal colonies are not important.

2.  Preparation of  E. coli strains
         a.  Separately grow the donor strain and the mobilizer strain to exponential phase in LB containing the 
         appropriate antibiotics.     
 
         b.  Based on the OD600 of the cultures, combine an equal amount of donor and helper cells in a centrifuge
         tube.  Centrifuge at a maximum of 2,000 x g for 10 min.  If the two strains were grown with different 
         antibiotics, a wash with plain LB is required before this step.

         c.  Decant the supernatant and resuspend the pellet to a density of OD600 = 9 or 10 in LB.

3.  Conjugation procedure

         a.  Lay sterilized Millipore HATF 0.45 um filters on plates of AA  + 5.0 mM MOPS, pH = 7.8 + 2.5 mM 
         NH4Cl +   0.5%LB, solidified with 1% purified agar.

         b.  In a microfuge tube, combine 0.5 ml of the E. coli mix with 0.5 ml of the concentrated Nostoc, pellet
         for 30 sec in a microfuge. Decant most of the supernatant leaving behind about 150 ul of liquid in the tube.

         c. Resuspend the pellet very gently with a wide bore pipet tip and place the entire suspension on a filter 
         and spread evenly with a glass hockey stick.

         d.  Place the parafilm sealed plates in a 1% CO2 enriched (for most rapid growth) incubator at low light 
         intensity (2-3 W/m2) overnight.

         e.  Transfer filters to AA  +  5.0 mM MOPS, pH=7.8 +2.5 mM NH4Cl +1% purified agar plates (without LB). 
         Keep in low light for two more days then move to higher light (4-6 W/m2) for 2 days.  This allows time 
         for expression of antibiotic resistance and recovery from the conjugation process.

         f.  Transfer filters to plates containing the appropriate antibiotics (see below) and continue to incubate
         under high light for about two weeks. The background should die back and resistant colonies will grow.

4.  Selective antibiotic concentrations for Nostoc:
         Neomycin:  25 ug/ml
         Ampicillin:10 ug/ml
         Erythromycin:  10 ug/ml
         Chloramphenicol:  60 ug/ml 
         Streptomycin:  1 ug/ml

5.  To identify the site of transposition:  isolate genomic DNA from the mutant (see genomic DNA isolation 
     protocol), digest the DNA with an enzyme that doesn't cut within the transposon (see Wolk et al.  1991.  
     Proc Natl Acad Sci USA. 88:5355-5359 for suggestions), ligate the digested DNA fragments and transform 
     E. coli.  The recovered plasmid should contain the transposon plasmid and flanking genomic DNA 
     sequences.

6.  For gene replacement: the gene of interest is inactivated either by insertion or deletion and then cloned 
     into pRL271 (or a similar plasmid) containing SacB.  The first recombination event (integration) should give
     rise to colonies, which are chloramphenicol resistant (from pRL271), plus any antibiotic markers inserted into 
     the gene of interest. The second recombination event (resolution) will give rise to sucrose resistant colonies. 
     We typically use 5% sucrose. It is important to check the plasmid constructions in E.coli for sucrose 
     sensitivity prior to conjugation into Nostoc.  We verify genotypes by Southern hybridizations. Resolution
     frequencies are variable, thus we keep cultures growing for resolution until the desired mutant has been
     identified and isolated.