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

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 3

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. 6

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 7

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 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.
Purified hSSB1
PAGE gel
-ve
+ve 9

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