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Mark Fisher

hSSB1 Bacterial Transformation and Expression. Mark Fisher. Research immersion project 84 Experimental lead: Syed Ali Naqi. Project aims. Aim 1: Purify hSSB1 proteins. Aim 2: Demonstrate hSSB1 mutations effect on protein function. Project stages.

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Mark Fisher

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  1. hSSB1 Bacterial Transformation and Expression Mark Fisher Research immersion project 84 Experimental lead: Syed Ali Naqi

  2. Project aims • Aim 1: Purify hSSB1 proteins. • Aim 2: Demonstrate hSSB1 mutations effect on protein function.

  3. Project stages Stage 1: Transfection of bacterial cells with hSSB1 construct and culture into a larger population (Monday-Tuesday). Stage 2: Induction of hSSB1 expression and purification (Tuesday). Stage 3: Demonstrate successful transformation, expression and purification of hSSB1 (Wednesday – Thursday). * Time lines and procedures may change

  4. Project overview Transfection Selective Culture Induction IPTG Bacterial genome Bacterial cells Plasmid hSSB1 Confirmation Purification Ni 2+ Ni 2+ Ni 2+

  5. hSSB1 construct (plasmid) Protein coding gene (Expressed with IPTG) • Plasmids • Small circles of DNA bacteria exchange with each other. • We inserted hSSB1 gene into a commercially available plasmid. Kanamycin resistance gene (always on) hSSB1 construct hSSB1 Protein Normal hSSB1 sequence Hexa histidine-tag

  6. Stage 1 • Transfect E. coli (BL21) cells with mutant plasmids. • Inoculate selective media with transformed cells and allow them to grow. • Because the media contains an antibiotic only those with the plasmid will survive. • Pick colonies for further culture. • Grow in LB overnight.

  7. Stage 2 IPTG • Grow cells to an ideal density and induce hSSB1 expression with IPTG. • Purify hSSB1 • Pellet bacterial cells with centrifuge. • Lyse cells with IGEPAL and sonication. • Pellet cell detritus with centrifuge. • Purify hSSB1 from above supernatant with nickel beads. Sonication and detergent

  8. Nickel bead purification hSSB1 & other bacterial proteins. Imidazole 300 mM Ni 2+ • Imidazole disrupts intermolecular interactions between his-tag and nickel beads. • Purified protein flows through in eluent. Wash Ni 2+ Ni 2+ Other bacterial proteins don’t interact with the beads. Eluent

  9. Stage 3 Purified hSSB1 • Confirm hSSB1 has been properly expressed and purified by • Polyacrylamide gel (PAGE) • Separate by size of protein. • Electrophoretic mobility shift assay (EMSA) • hSSB1 (mutants + wt) and DNA fragments are allowed to interact then run on a gel. • Separation by size and interaction between protein and substrate. PAGE gel -ve +ve

  10. ? Confused? ? ? Wash ? Transfection ? Ni 2+ Immidazole Eluent ? ? ? We will cover all this and more in greater detail as we go

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