Construction and Characterization of purD Gene Deleted Brucella abortus
Construction and Characterization of purD Gene Deleted Brucella abortus
Muhammad Ilyas Riaz1, Masood Rabbani1*, Sohail Raza1, Ali Raza Awan2, Aleena Kokab1 and Iqra Nazir3
Construction and confirmation of pUC19-K-UP-DN gene deletion cassette. A, Illustration of map of pUC19-K-UP-DN gene deletion plasmid cassette; B I, Amplification of KM fragment. Line M showed 1Kb ladder; Line 1,2,3 showed 1004 bp KM amplicons; B II, Restriction digest analysis of pUC19-K. Line M showed 1Kb ladder; Line 1,2,3 showed transformed E.coli DH5α cells having 2686 bp band of pUC19 and 986 bp band of KM; B III, Amplification of UP fragment. Line M showed 100bp ladder; Line 1,2,3 showed 492 bp UP amplified amplicons; B IV, Restriction digest analysis of pUC19-K-UP. Line M showed 1Kb ladder; Line 1,2,3 showed transformed E. coli DH5α cells having 2686 bp band of pUC19 and 1455 bp band of K-UP fragment; B V, Amplification of DN fragment. Line M showed 100bp ladder; Line 1,2,3 showed 482 bp DN amplified amplicons; B VI, Restriction digest analysis of pUC19-K-UP-DN. Line M showed 1Kb ladder; Line 1,2,3 showed transformed E.coli DH5α cells having 2686 bp band of pUC19 and 1909 bp band of K-UP-DN fragment.
Confirmation of the ΔpurD gene deleted Br.abortus mutant. A, PCR for Br.abortus (498 bp). Line M showed 1Kb ladder; Line 1 1st Br. abortus transformed colony DNA; Line 2 2nd Br. abortus transformed colony DNA; Line 3 is the positive control. B, PCR for KM confirmation (986 bp). Line M showed 1Kb ladder; Line 1 ΔpurD deleted mutant DNA; Line 2 is the positive control. C, PCR for detection of purD gene (1280 bp). Line M showed 1Kb ladder; Line 1 ΔpurD deleted mutant DNA; Line 2 is the positive control.
Growth kinetics comparison of the ΔpurD mutant and RB51 strain. O.D values at different time intervals when grow in non-enriched TSB medium without supplementation of adenine, guanine, thiamine and hypoxanthine. A, and in TSB enriched medium with supplementation of adenine, guanine, thiamine and hypoxanthine. B, Statistically significant differences in growth curve between parent RB51 strain and ΔpurD mutant was determined by using two-way ANOVA (***, P <0.05).
