Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit

Figure 4

Successful in locus tagging of L. donovani CK1.1 with mNG-myc tag. (a) Proteins were extracted from LdB pTB007 or LdB CK1.1-mNG-myc promastigotes in logarithmic phase and twenty micrograms was analysed by Western blotting using an anti-Myc tag antibody (Top panel). The Coomassie-stained membrane of the blot is included as a loading control (bottom panel). Protein weight in kDa is indicated on the left. The expected size of the fusion protein is 69,8 kDa. The lower band indicated with an asterisk () may be a result of protein degradation. (b) Promastigotes were seeded at promastigotes/mL and cultured for 7 days, and aliquots were taken every 24 h for analysis. Cell number (black symbol) and mNeonGreen fluorescence intensity (white symbol) were assessed by flow cytometry in triplicate in two independent experiments. Fluorescence intensity of the LdB pTB007 strain was used for normalization. Cell lines: LdB pTB007 (circle) and LdB pTB007 CK1.1-mNG-myc (diamond). (c) Similar to (b), except that promastigotes were seeded at promastigotes/mL, shifted to 37°C and pH5.5 and cultured for 6 days. (d) Similar to (a), except that proteins were extracted from LdB pTB007 or LdB CK1.1-mNG-myc axenic amastigotes (48 h after temperature and pH shift).

(a)

(b)

(c)

(d)