# Implementation Studies of Bacteriophage \(\lambda\) O
## 2 Results
### Implementation for \(O\) Activity in Trans  
The wild type \(O\) protein is 299 amino acids (AA) [43]. Alleles of \(O\) were cloned into an expression plasmid, Figure 3. Each allele was from an RK\({}^{-}\) mutant for which complementation analysis had suggested was \(O^{+}\).  
Each plasmid was transformed into 594 cells, creating strains as 594[pclpR-\(O\)-timm] that were used as hosts for \(\lambda\) Oam plating. The assays for \(O\) complementation were incubated at 30, 37, 39 and 42 \({}^{\circ}\)C. The results for plating assays at 42 \({}^{\circ}\)C, where the \(O\) allele is fully expressed, are shown in Table 2. Phages whose \(O\) allele produced 76 or 294 AA's of O were weakly complemented by O\({}^{+}\), whereas the Oam905 mutation expressing 37 AA of O was not complemented by O\({}^{+}\). Complementation was improved 5- to 6-fold in two of the three \(\lambda\) Oam suppression assays by the addition of a SPA tag to the COOH end of the wild type \(O\) sequence, i.e., the addition of a seven AA linker (GGSGAPM) joined to the 69 AA SPA tag [47] sequence. The _O-ori_:98 mutation removing ITN4 within \(O\) was incapable of complementing for O. Remarkably, \(O\) alleles with the _ori_:ti12 point mutation in ITN4 and those with _ori_:95 and _ori_:96 in-frame deletions, respectively, of 12 and 15 bp's within the High AT-rich region of _ori_\(\lambda\), were each able to complement all three Oam mutants. Any condition where P, or a portion of the N-terminal end of P was expressed, completely prevented O-complementation. Induced prophage strains with insertions within \(P\), but with sequenced intact \(O\) genes that would be fully derepressed when shifted to 42 \({}^{\circ}\)C were incapable of providing for O complementation. The RK\({}^{-}\) ilr mutants, previously designated phenotypically as \(O^{+}\)[11], proved to have insertions or deletions in \(O\) and should not complement, as was found for the cloned prophage \(O\) genes from mutants 208b, 223a and 541c (each of which could complement for \(P\)[36]).  
Figure 3: Expression vector pclpR-(\(O\) alleles)-timm. Expression of gene \(O\) or an allele occurs upon inactivation of the CI repressor by raising cells grown at 30 \({}^{\circ}\)C to 42 \({}^{\circ}\)C. Immediately following the 299 codons of \(O\) is an ochre stop codon, where the last base in TAA represents the first base of the _ClaI_ restriction site ATCGAT.  
\begin{table}
\begin{tabular}{l c c c} \hline \hline  & \multicolumn{2}{c}{**EOP of A Phage with Oam Mutants b **} \\ \cline{2-3}
**Host Strains and [Plasmid] \# * & \(\lambda\)_d_857 & \(\lambda\)_d_857 & \(\lambda\)_d_857 \\  & **Oam905 c ** & **Oam29 c ** & **Oam29 c ** \\  & **(37 AA of O)** & **(76 AA of O)** & **(294 AA of O)** \\ \hline Pm* _SupE_4  & 1.0 & 1.0 & 1.0 \\ Pm* _SupP_  & 0 & 0 & 0 \\ Complementation by O variations & & & \\ O* combinations & & & & \\
[O]* + 465 & 0 & 0.2 & 0.1 \\
[O-SPA] \({}^{\dagger}\), p472 & 0 & 1.0 & 0.6 \\
[ope-OI] \(\,\)s, p677 & 0.05 & 0.1 & 0.1 \\ O null mutations h  & 0 & 0 & 0 \\
[O-ii/2208d], p488 & 0 & 0 & 0 \\
[O-ii/223d], p486 & 0 & 0 & 0 \\
[O-ii/541c], p485 & 0 & 0 & 0 \\ O-origin (ii/n) mutations l  & & & & \\
[O-ori:98], p489 & 0 & 0 & 0 \\
[O-ori:95], p491 & 0.2 & 0.3 & 0.2 \\
[O-ori:96], p492 & 0.3 & 1.0 & 0.3 \\
[O-ori:112], p493 & 0.3 & 0.1 & 0.3 \\ O-P combinations l  & & & & \\
[O-36P], p565 & 0 & 0.01 & 0 \\
[O-63P], p566 & 0 & 0 & 0 \\
[O-P], p569 & 0 & 0 & 0 \\
[ope-O-P] k567, p568 & 0 & 0 & 0 \\ \(RK^{-}\) - O* P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-