Fig 1.
Concept for ORF selection using split beta-lactamase protein fragment complementation-based vector system pVMAKORF001 in E. coli.
Depending on the frame and solubility of the fragment cloned between two parts of beta-lactamase, it can have two fates. (A) If the cloned fragment encodes an in-frame protein, it will reconstitute beta-lactamase activity and will grow on ampicillin containing media plate depending on its solubility. (B) If the cloned fragment encodes an off-frame protein, it will not produce full-length beta-lactamase and the clone will be eliminated upon selection on ampicillin containing media plate.
Fig 2.
Schematic representation of pVMAKORF001 vector.
Only relevant genes and restriction sites are shown. The map is not to scale. Kanr, kanamycin resistance gene; ColE1, origin of replication; Fori, phage M13 origin of replication; AraC and pBAD, Cassette encoding arabinose promoter and its regulator AraC; BlaSS, Native bla signal sequence; α 24–195, Bla-Alpha fragment encoding 24–195 amino acids of beta-lactamase; M182T, Methionine 182 to threonine mutation; NGR, Asparagine-Glycine-Arginine tripeptide; S1 and S2, (Gly4Ser)3 linker; Sp. Spacer sequence; TEV, Tobacco Etch Virus protease sequence; ω 196–286, Bla-Omega fragment encoding 196–286 amino acids of beta-lactamase; SacB and SacR, gene cassette encoding levansucrase of B. subtilis. (A)-(C) Sequence of different cassettes of the vector. (D) Sequence of insert to be cloned in the vector. (E) Insert carrying 4 base 5’ overhangs after T4 DNA polymerase treatment in the presence of dTTP.
Fig 3.
ORF selection profile of model constructs upon selection on increasing concentrations of ampicillin.
Clones encoding in-frame Alpha-Spacer-Omega, Alpha-19kDa-Omega, and off-frame Alpha-stop proteins were pre-induced with 0.0002% arabinose, and serial dilutions were spotted on plates containing LB media with 0.0002% arabinose and increasing concentrations of ampicillin (0 μg/ml to 100 μg/ml).
Table 1.
Efficiency of ORF selection with a culture mix mimicking ORF to non-ORF ratios observed with DNA fragment libraries.
Fig 4.
Analysis of ORF selection efficiency by high-throughput sequencing of MTBLIB42 before and after ORF selection.
MTBLIB42C01 and MTBLIB42C02 libraries were sequenced using MiSeq Nano v2 chemistry for 2 x 250 cycles. A total of 5,66,496 (MTBLIB42C01) and 5,38,956 (MTBLIB42C02) merged reads aligned to 30 M. tuberculosis genes, and the data was further analyzed using in-house developed pipeline, ORFSELECT. (A) MTBLIB42C01, (B) MTBLIB42C02. However, the number of in-frame clones in the unselected library was higher (42.3%) as compared to the theoretically possible number (5.6%; 1 in 18 is in-frame). To further investigate this difference, we generated overlapping 30 gene fragment libraries of 100 bp, 200 bp and 300 bp fragment size with 1 bp incremental shift in silico and determined the number of theoretically possible in-frame clones. The number of in-frame clones for theoretical 30 gene fragment libraries of size 100 bp, 200 bp and 300 bp was found to 58.3%, 40.9%, and 31.9%, respectively, which corroborated with our experimental findings.
Fig 5.
Expression and solubility analysis of 12 randomly selected clones obtained after transfer of MTBLIB42C02 in pVMH10D-BAP001 expression vector.
Twelve randomly selected clones were subjected to auto-induction at 18°C and solubility was assayed using PopCulture assay. The total cell (T) and soluble (S) fractions were analyzed using SDS-PAGE.