LIBRARY CONSTRUCTION AND SCREENING

Partial libraries of size-selected DNA fragments from endophyte strains ES (E. typhina) and Lpl (LpTG-2) were constructed in the vector M 1 3mp 1 9 (Section 2. l .3) and screened using methods described by Sambrook et al. ( 1989).

2.12.1 CALF ALKALINE PHOSPHATASE (CAP) TREATMENT OF VECTOR

Digested vectors were treated with calf alkaline phosphatase (CAP) to prevent self ligation of vector in subsequent ligation reactions. Vector DNA, 5 )lg, was digested to completion in an excess of the appropriate restriction enzyme (Section 2.7). The restriction enzyme was inactivated by heating for 10 min at 65°C and 0.5 U of CAP was added and incubated at 37°C for 30 min. Na2EDTA (pH S.O) to a final concentration of 5 mM, SDS to give a final concentration of 0.5%, and proteinase K to a final concentration of 50 )lglml were added, and the mixture was incubated at 56°C for 30 min . The DNA was purified b y sequential extractions with equal volumes of phenol, phenol/chloroform ( 1 : 1 , v:v), and chloroform with centrifugation for 5 min at 1 5,000 x g between steps. DNA was then precipitated by addition of 1110 volume of 3 M sodium acetate (pH 7.0) and 2.5 volumes of 95% ethanol, chilling at -20°C for 30 min and centrifugation for 10 min at 1 5 ,000 x g. The pellet was washed in 70% ethanol, air dried, resuspended in MilliQ water to a concentration of 20 ngf)ll, and stored at 4°C.

2.12.2 PREPARA TION OF LINKERS

BamHI linkers were constructed from the self annealing of 51-chemically phosphorylated 51-pCGGGATCCCG oligonucleotides (Life Technologies) that were based on a sequence by Stratagene. In a microcentrifuge tube, 2 III of 1 0 x annealing buffer ( 1 00 mM Tris-HCl (pH 7.6), 1 0 mM Na2EDTA, and 1 M NaCI), 4 nmol of oligonucleotide, and MilliQ water to a total volume of 20 III was mixed. The tube was incubated in a small waterbath at 65°C for 10 min, then the waterbath turned off and allowed to cool slowly to room temperature. The annealed Iinkers, at a final concentration of 1 00 pmol/lll, were stored at _20DC.

2.12.3 PREPARATION OF LINKERED INSERT DNA

Genomic inserts were prepared by the digestion of genomic DNA using combinations of the blunt end 4 bp cutters AluI, RaeIII, and ThaI, as well as BamHI (Section 2.7). Digests were separated on SeaPlaque gels in T AE buffer (Section 2.8), and fragments in the size range 200 to 500 bp (E8 digests), or 1 00 to 1 000 bp (Lp1 digests) were excised and purified from agarose (Section 2.5. 10). Linkers were ligated to the blunt ends of the inserts to facilitate cloning, and the ligation reaction was set up on ice as follows: 100 to 500 ng of blunt end DNA fragments, 1 to 2 ug of phosphorylated linkers (Section 2. 1 2.2), 4 III of 5 x ligation buffer (Section 2.4.4), 0.8 U of T4 DNA ligase (New England Biolabs), and MilliQ water to a total volume of 20 Ill . Ligation was canied out for 1 6 to 20 hr at 4°C and the ligase was inactivated by heating the reaction to 65°C for 1 5 min. Half microlitre aliquots of the ligation reaction were taken before addition of the ligase and after the ligation reaction and run on a mini gel (Section 2.8.2) to check for ligation. If fragments appeared ligated, 10 III of the appropliate 10 x restriction enzyme buffer, 20 to 50 U of restriction enzyme and Mil l iQ water to a total volume of 1 00 III was added to the inactivated ligation reaction and incubated for at least 4 hr at 37°C. A fUl1her 1 0 U of restriction enzyme was added and the reaction incubated for another hour, then Na2EDTA (pH 8 .0) was added to a final concentration of 1 0 mM to stop the reaction. DNA was extracted with an equal volume of phenollchloroform ( 1 : 1 , v : v) and

centrifuged for 2 min at 1 5,000 x g and the aqueous phase extracted. The linkered DNA fragments were purified from the digested linkers by gel purification in SeaPlaque agarose gels in TAE buffer as outlined in Section 2.5. 10.

2.12.4 LIGATION REACTIONS

Ligation reactions were performed in 1 0 or 20 fll volumes. The quantities of reagents for a 20 fll reaction are as follows: 4 fll of 5 x ligation buffer (Section 2.4.4), 20 ng of vector, 40 U of T4 DNA ligase, and a 2 to 5 molar excess of insert vector DNA. The ligation mixture was incubated for at least 1 6 hI' at 4°C. To check that ligation had occurred, two 2 fll samples of the ligation mixture were removed, one before adding the ligase and the other after ligation, mixed with SDS loading dye (Section 2.4.7) and separated on a mini gel (Section 2 . 8.2).

2.12.5 PREPARATION OF ELECTROCOMPETENT XL-1 CELLS

One litre of LB (Section 2.3. 1 ) was inoculated 11100 with an overnight culture of XL- 1 cells (Section 2.2.4) and grown at 37°C with vigorous shaking to mid-log phase (A6oo of 0.5 to 1 .0, approximately 3 hr). The cells were chilled on ice for 20 min and harvested by centrifugation at 4,000 x g for 1 0 min at 4°C. The cells were resuspended twice in ice­ cold water ( 1 litre, then 500 ml ,), followed by resuspension in 20 ml ice-cold 1 0% (v/v) glycerol, centrifuging as above between steps. The cells were resuspended in 4 ml ice­ cold 1 0% (v/v) glycerol and stored in 40 fl l aliquots at -70°C.

2.12.6 TRANSFORMATION BY ELECTROPORA TION

Aliquots, 40 fll , of electrocompetent cells (Section 2. 1 2.5) were thawed gently at room temperature, placed on ice and 1 to 2 fll of DNA to be transformed was added. The tube contents were gently mixed, and tubes were left on ice for 1 min. The Gene Pulser (Bio­ Rad) was set to 25 flF and 2.5 kV, and the pulse controller set to 200 ohms resistance in parallel with the sample chamber. The mixture of DNA and cells was transferred to an

ice cold 0.2 cm electroporation cuvette and pulsed at the above settings. The time constant was checked and, if this was between 4 and 5, the cells were immediately resuspended in 1 ml of SOC medium (Section 2.3.3). Transformed cells were incubated at 37°C for an hour to aid the recovery of the E. coli. A positive control of 2 ng pUC l 1 8, and a negative control of water only was always employed with each set of transformations. Cells were plated (Sections 2.2.4 and 2.2.5) at suitable dilutions onto selective LB plates (Section 2.3 . 1).

2.12.7 FILTER LIFTS AND PRIMARY LIBRARY SCREENING

Recombinant M 1 3mp 1 9 phage were plated at approximately 1 0,000 plaques per plate to form the primary library. Filter lifts were taken from the library by marking nylon filters (Hybond N+, Amersham) asymmetlically and placing on the phage plates for 1 min . The marks were transferred to the plates for later alignment. Filters were placed on blotting paper to air dry and DNA was cross-linked by placing face down on a UV transilluminator and exposing for 2 min. To eliminate false positives, two lifts were taken from each plate where the second lift was left on the plate for 2 min . Hyblidisation was performed as described in Section 2 . 1 1 . Positive plaques, identified by alignment of the plate with positive hybridisation signals from the duplicate plaque lifts, were picked with a sterile cut-off 1 ml tip. The agar plug was ejected into 1 ml of LB medium (Section 2.3 . 1 ) and stored at 4°C overnight to elute the phage from the agar.

2. 12.8 SECOND ROUND LIBRARY SCREENING

Phage from first round positive plaques (Section 2. 1 2.7) were diluted to 1 0-6 to 1 0-8 in TE buffer ( 10: 1 ; Section 2.4. 1 2) and 100 III plated as previously described (Section 2.2.5). Plates containing between 30 and 300 plaques were lifted and hybridised as previously described (Section 2. 1 2.7). Positive plaques from the second round of library screening were picked with a toothpick and used to inoculate liquid cultures (Section 2.2.5).

2.12.9 SCREENING POSITIVE CLONES

Unique clones were identified by digesting plasmid DNA, isolated from positive plaques using a rapid boiling method (Section 2.5 .5), with BamHI (Section 2.7) to excise the insert fragments. Digests were separated on mini gels (Section 2 . 8.2) and unique clones were identified based on the size of their inserts. If many clones had inserts of the same size, they were analysed using a 'single tract sequencing' approach, where sequencing reactions were carried out using only a single terminating dideoxynucleotide. This was carried out as described in Section 2. 1 5 . 1 but reactions contained the following quantities: 2 to 20 fmol of template DNA, 0.8 III 10 x cycling mix, 4 pmol of sequencing primer, 0.2 111 of [a_33p] _{dCTP (�2,500 Ci/mmol, Amersham), 2 111 termination mix and} MilliQ water to bring the volume to 8 Ill. Unique clones were then fully sequenced (Section 2. 1 5).