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Bacterial Transformation and Expression of hSSB1 Proteins Research Project

This research immersion project led by Mark Fisher and Syed Ali Naqi aims to purify hSSB1 proteins and demonstrate the effect of mutations on protein function. The project involves transfection of bacterial cells with hSSB1 construct, culture, induction of protein expression, and purification stages. Using techniques like IPTG induction and Ni2+ bead purification, the team will confirm successful transformation and expression of hSSB1, followed by protein analysis through gel electrophoresis and EMSA. Stay tuned for detailed procedures and results.

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Bacterial Transformation and Expression of hSSB1 Proteins Research Project

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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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