Directional Cloning into pEX-2 with a Dam-Blocked Site and High Background
The 1.4 kb open reading frame of a bacterial lipase is to be cloned into the 5.2 kb expression vector pEX-2 (ampicillin resistance, ColE1 origin). The multiple cloning site contains unique sites in the order EcoRI, KpnI, BamHI, XbaI, HindIII; a lac promoter lies upstream of the site and a His6 tag downstream of HindIII. The gene contains a single internal EcoRI site at position 900 and a single internal BamHI site at position 350, but no KpnI, XbaI or HindIII sites. The insert will be made by PCR from genomic DNA, so tails can be added. Plasmid pEX-2 is routinely prepared from E. coli DH5α, which is dam⁺. In this vector the XbaI recognition site lies immediately downstream of the BamHI site (context 5'-GGATCTAGA-3'). A pilot ligation of doubly cut vector with doubly cut insert gave 40 transformants on ampicillin; 31 of them produced only a 220 bp product in colony PCR with primers flanking the cloning site.
1. Select the two restriction sites to use and justify rejecting each of the alternatives, including the methylation issue.
Read the worked answer
Eliminate by inspection first:
- EcoRI - cuts inside the gene at 900 bp, so it would split the ORF.
- BamHI - cuts inside the gene at 350 bp, same problem.
- XbaI - absent from the insert, but in this vector the site sits in the context 5'-GGATCTAGA-3', which contains a Dam target (GATC). DH5α is dam⁺, so the adenine is methylated and XbaI cannot cut. Choosing XbaI would give vector cut at only one end.
- KpnI and HindIII - both unique in the vector and absent from the insert.
So the pair is KpnI + HindIII. They produce non-identical, non-compatible ends (KpnI leaves a 3' GTAC overhang, HindIII a 5' AGCT overhang), which forces orientation and prevents insert self-ligation in the wrong direction. The insert should be amplified with a KpnI tail at the 5' end and a HindIII tail at the 3' end, placing the ORF in frame with the downstream His6 tag. If XbaI were essential, the vector would have to be prepared from a dam⁻ strain such as JM110.
2. For 100 ng of 5.2 kb vector, calculate the mass of 1.4 kb insert needed for a 3:1 insert:vector molar ratio, and state what would go wrong at a 20:1 ratio.
Read the worked answer
Moles are proportional to mass/length, so for equal masses the vector is 5.2/1.4 = 3.71 times more concentrated in molecules than the insert.
Insert mass = vector mass × (insert length/vector length) × (molar ratio) = 100 ng × (1.4/5.2) × 3 = 100 × 0.269 × 3 = ~81 ng.
Check: 100 ng vector = 100/5200 = 0.0192 pmol-equivalent units; 81 ng insert = 81/1400 = 0.0579 units, a ratio of 3.0. Consistent.
At 20:1 the insert would be ~540 ng. Excessive insert favours intermolecular insert–insert ligation and multiple inserts per vector, and it raises the chance of a vector receiving two insert molecules in tandem, which would shift the reading frame relative to the His6 tag. It also raises the background of ligated insert concatemers that can be taken up and give false positives in colony PCR. A ratio of 1:1 to 3:1 is the usual working range.
3. Interpret the colony PCR result (31/40 clones give 220 bp; recombinants should give ~1.62 kb) and propose two concrete changes to raise the proportion of recombinants.
Read the worked answer
The 220 bp product is the empty vector, the size expected from primers that flank an unoccupied cloning site. A recombinant adds 1.4 kb, so it should give about 220 + 1400 = 1.62 kb. With 31/40 (78%) empty, the ligation is dominated by recircularised vector.
The most likely cause is incomplete cutting at one of the two sites. A vector cut at only one end still recircularises efficiently by intramolecular ligation, losing a few bases but retaining the primer-binding regions, so it scores as empty. Undigested plasmid carried through the cleanup does the same. If the pilot used XbaI, Dam methylation would explain it directly.
Fixes: (1) treat the doubly digested vector with alkaline phosphatase (SAP/CIP) so that any singly cut or uncut molecules cannot self-ligate, while the insert supplies the 5' phosphates; (2) gel-purify the 5.2 kb doubly cut band and run a small analytical digest to confirm both sites are cut before ligating, and reduce the vector mass in the reaction. Screening more colonies with a second, insert-internal primer would also catch clones that carry a truncated insert.