Hydoxylamine Mutagenesis of Plamid DNA
via Peter Ames
0.5M Potassium phosphate, pH 6.0, 5 mM EDTA. Add 0.5M K2HPO4 (about 10- 20 mls) to 100 ml 0.5M KH2PO4 until the pH increase to 6.0.
Hydroxylamine. In a large plastic Falcon tune combine 0.35g NH2OH, 0.56ml 4N NaOH, and water to 5mls and mix well to dissolve. Prepare another tube with a control solution of NaOH and water but leave out the hydroxylamine.
10mM Tris, pH7.5, 1mM EDTA (for dialysis). Prepare 2x2l in wide-mouth flasks and cool them to 4oC.
In small plastic Falcon tubes, mix 0.4ml of 0.5M KPO4, pH 6.0, 5mM EDTA stock, 0.8ml of ddH2O, and 0.8ml of the Hydroxylamine stock. Prepare two tubes for each plasmid. Also prepare a matching control tube for each of the above tubes, containing 0.4ml 0.5M KPO4, 5mM EDTA stock, 0.8ml of ddH2O, and 0.8ml of the no hydroxylamine stock.
Add 2ug of plasmid DNA to be mutagenized into each of the above tubes, and incubate one pair for 60 min and the other for 90 min at 70oC.
Transfer the solutions to dialysis tubing the dialyze vs 10mM Tris, pH7.5, 1mM EDTA at 4oC for 2-3 hr. Then change the buffer and dialyze for another 2-3 hr. NOTE: It is critical to recovery of the plasmid DNA to do this dialysis before ethanol precipitation.
After dialysis, transfer each solution into a large (12 ml) plastic Falcon tube, and decrease the volume to about 400ul by dehydrating with a total of about 12 ml of anhydrous isobutanol. Transfer the 400ul of plasmid containing solution to 1.5ml Eppendorf tubes, and ethanol precipitate by adding 0.1 vol of 3M sodium acetate, pH 3.0, followed by 2.5 vol of ethanol. Leave on dry ice until frozen, spin for 10 min in the microfuge to pellet DNA, rinse the pellet once with 70% ethanol, spin it again and then dry it.
Resuspend the DNA pellet from each tube in 50 ul of sterile ddH2O, and use
5 ul of this to retransform your host of choice. Expect 4-5,000 colonies per plate from 10% of untreated controls, and 10-50% of this number for the 60 min and 90 min treated samples respectively.
Efficacy of the mutageneis protocol. Mutagenesis of pPA56. I originally developed the above mutagenesis protocol to mutagenize plasmids encoding K1 and R1 domains of Tsr. Due to the underwhelming results from expression of these plasmids in Che+ hosts, this is now on hold. However, to show the effectiveness of the mutagenesis procedure, I used it to mutagenize pPA56. 2ug of pPA56 was mutagenized for 60,90, and 120 min at 70C, and the mutagenized plasmids were used to transform RP5700. Treatment for 60,90, and 120 min resulted in 50%, 85%, and 90% reductions, respectively, in the number of ampicillin resistant transformants. I screened 200 transformants from each mutagenized sample, and 200 from each control (treated for the same time but without hydroxylamine) for chemotaxis on T-swarm plates containing ampicillin, or ampicillin plus IPTG at 1mM (to induce pPA56). No mutants were found, i.e. all colonies were Che+ without induction and Che- with induction, from the control samples. 13/200 (6.5%), 8/200 (4%), and 5/200 (2.5%) of the colonies from the 60, 90, and120 min time points, respectively, displayed swarming behavior different from that of the parental plasmid. Of the 26 total mutants, 3 resulted in constitutive expression of the Tsr fragment (Che-, swarms +/-IPTG), 7 were Che+ in the presence of absence of induction (proably knockouts), 12 exhibited induced swarms which were larger in size than that of pPA56 (partial function?), and 4 appeared to be smaller than swarms of induced pPA56 (enhanced CW or CCW?). Examination of induced cell lysates by SDS-PAGE showed that 4/7 potential knockouts produced no apparent fragment protein, although the remaining 3 expressed pretein of the same size and similar quantity to that from pPA56. 5 of the mutants with reduced inhibition also expressed protein like that from pPA56.
Last Update: Thursday, 19-Jun-2014 12:02:26 PDT
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