To construct the BAC library for strain Lpha5T, genome DNA of Lpha5T was first partially digested into suitable size using restriction enzyme HindIII. However, no partial digested fragments were generated (Figure 4.1), likely due to the high G + C content of genomic DNA of Lpha5T. Thus, to investigate its restriction profile, the Lpha5T genomic DNA was further digested with several other restriction enzymes, including BamHI, EcoRI, HindIII, MspI, SphI, XbaI, RsaI, PstI, NotI, and NdeI. The results showed that Lpha5T genomic DNA could be digested into smear by restriction enzymes BamHI, MspI, SphI, PstI, NotI, and NdeI, whist no obvious digestion was observed by restriction enzymes EcoRI, HindIII, XbaI, and RsaI. The digested profile of the Lpha5T genome DNA was summarized in Table 4.1.
To predict the potential protein-coding regions, analysis of ORFs was performed. The 2,186 bases sequence was subjected to DNAStar software to find the ORFs. Firstly, ORFs, which consisted of sequences longer than 50 sense codons starting with ATG were searched. In total, five ORFs were found within the sequence (if two ORFs overlapped on either strand, the longer one was chosen). They were designated as ORF1, ORF2, ORF3, ORF4, and ORF5. The number of nucleotide contained in each ORF was 84, 138, 507, 321 and 265 bp, respectively. All the five ORFs were translated into amino acid sequences. Each of the five ORFs encoded a peptide with a length of 27, 45, 168, 106 and 88 amino acids, respectively. Further, the five ORFs were analyzed to determine their specific features, including the nucleotide range in the fragment, molecular weight, %G+C, number of amino acids encoded, number of strongly basic(+) amino acids (K,R), strongly acidic(-) amino acids (D,E), hydrophobic amino acids (A,I,L,F,W,V) and polar amino acids (N,C,Q,S,T,Y) encoded by the ORFs. The G+C content of the five ORFs was 60.71%, 73.56%, 72.98%, 66.98% and 66.79%, respectively. ORF3, ORF4, ORF5 contained amino acids that were strongly basic, strongly acidic, hydrophobic and polar amino acids, whilst ORF1 and ORF2 lacked strongly acidic amino acids. The features of the five ORFs were summarized in Table 4.2.