Electroporation and Plating of Geobacter Sulfurreducens

Aklujkar, Muktak 1-4

Electroporation and Plating of Geobacter sulfurreducens

1. Put sufficient sleeves of Petri plates in a glove bag where you have reserved sufficient space in a container for the next two weeks. a. Remember to open the sleeve and tape it shut b. Label it with the date it was put in the glove bag 2. Make sure that the glove bag has enough: a. Screw-cap microfuge tubes b. Loosen the caps but don’t open them completely c. Sterile spreaders d. Sterile blunt yellow tips e. Kimwipes f. Sharps disposal capacity. g. Two waste containers, for liquids and solids. 3. Make or borrow the anaerobic antibiotic stock solutions you will need. 4. To add 1 ml to each 100 ml serum bottle of plating medium. a. This corresponds to: 100X Stock Final concentration 1 mg/ml chloramphenicol 10 ug/ml 20 mg/ml kanamycin 200 ug/ml 40 mg/ml streptomycin 400 ug/ml 7.5 mg/ml spectinomycin 75 ug/ml (250 to 2000 ug/ml for plasmids) 2 mg/ml gentamicin 20 ug/ml 5. To make 25 ml of stock: a. Bung and crimp a small serum bottle b. Autoclave the bottle c. Filter the antibiotic into the bottle. i. DO NOT AUTOCLAVE ANTIBIOTICS. THEY WILL DEGRADE. d. Reduce a gassing station with H2 e. Flush for 15 minutes with N2 only i. CO2 will mess up the pH of your solution. f. Sterilize the bung of your serum bottle of antibiotic stock g. Insert two needles crossing diagonally, so that one is submerged in the solution and the other is in the headspace h. Attach a canula with N2 flowing to the submerged needle and prop up your serum bottle so that the solution doesn’t get into the other needle. i. Gas out for 30 minutes. 6. Your DNA should be concentrated a. But 1 ug/ul is too concentrated. 7. For crossover with the chromosome, 500 bp of homology is recommended. 8. Plan your experiment so that cells electroporated with plasmids will have 5 hours to recover between zapping and plating; 18 hours are recommended for crossover with the chromosome. 9. Label one pressure tube of 10 ml NBAF for each electroporation and one for the negative control (the host strain electroporated with no DNA). Aklujkar, Muktak 2-4

10. Anaerobically add 0.1 ml of 100 mM cysteine and 0.2 ml of 5% yeast extract to each tube. a. Ching recommends 0.4 ml of the cysteine, but no more. 11. When tubes are clear, thaw the electrocompetent cells on ice. 12. Thaw the DNA and chill it on ice. 13. Chill electroporation cuvettes on ice. 14. Chill some sterile blunt yellow tips if you want to be extra-nice to your cells, but the regular (sharp, unchilled) tips work just fine. 15. Go to the electroporation area with: a. P-2 pipettor b. P-200 pipettor c. P-1000 pipettor d. Tips e. Cells on ice f. DNA on ice g. Cuvettes on ice h. The reduced pressure tubes i. Sterile syringes attached to needles j. Aerobic phosphate-buffered NBAF k. Ethanol for swabbing. 16. Arrange items along the windowsill and bench in order of use, from the P-2 and tips on your right to the recovery tubes, syringes and needles on your left. 17. Set the BIO-RAD GenePulser to 1.47 in manual mode. 18. Turn on the Bunsen burner. 19. With the P-2, add 1-2 ul of DNA to a shot of cells a. Nothing for the negative control 20. Transfer cells and DNA to the cuvette with the P-200 21. Place Cuvette in the BIO-RAD GenePulser 22. Zap immediately to electroporate. 23. Aseptically take 1 ml of phosphate-buffered NBAF to the cells with the P-1000 24. Quickly draw them into a syringe. 25. Sterilize the recovery tube with Ethanol 26. Add the cells to recovery tube. 27. DISCARD THE SYRINGE AND NEEDLE IN A SHARPS CONTAINER. 28. Clean up the electroporation area and make sure that there are no needles in the regular trash. 29. Incubate your recovery tubes at 30oC for the period recommended above. 30. Meanwhile, melt sufficient serum bottles of NBAF agar in the autoclave for 12 minutes. 31. Cool the agar in the 55oC bath in Rm. 406. 32. Make master mix: a. Take a serum bottle of 25 ml of 100 mM cysteine b. Anaerobically add: i. 10 ml of 5% yeast extract ii. 5 ml of 1% MgSO4 iii. 5 ml of 0.4% CaCl2 Aklujkar, Muktak 3-4

33. Anaerobically add 9 ml of master mix to each serum bottle of NBAF agar. a. Do not relieving the pressure so that the agar remains melted. 34. Add 1 ml of the appropriate 100X antibiotic solution. 35. Invert the serum bottle to mix it and take it into the glove bag. 36. Sterilize the work surface with ethanol 37. Keep the pliers sterile within an ethanol-soaked Kimwipe. 38. Sterilize the bung and crimp of the serum bottle with an ethanol-soaked Kimwipe 39. Use the pliers to remove the bung and crimp of the serum bottle. 40. Pour the plates and stack them until it is time to spread them. a. You will get 4 plates (same antibiotic) out of each 100 ml of NBAF agar b. Do not try to get more or the plates will be too dry. 41. In the glove bag, label sufficient screw-cap microfuge tubes 42. Aseptically transfer 1 ml of recovered cells to each tube with a syringe. 43. Screw the caps tightly 44. Take the tubes out of the glove bag 45. Spin them in the microfuge for one minute 46. Take them back into the glove bag. 47. Dump out most but not all of the liquid from each tube a. One quick inversion, no tapping 48. Resuspend the cells with a blunt yellow tip 49. Plate them with a sterile glass spreader 50. Incubate the plates in a container within the glove bag. a. Include paper towels in the container to absorb excess moisture. 51. Change the palladium and desiccator cartridges. 52. Colonies should take 4 to 11 days to show up, sometimes longer. a. Chloramphenicol and spectinomycin resistant colonies usually take 11 days to appear b. Background can be high, so always do a negative control! 53. Streak out at least 4 colonies of each mutant on selective plates. 54. After they grow up, disperse isolated colonies in screw-cap microfuge tubes of NBAF a. Sterile individually wrapped loops are in the glove bags. b. Make 1 ml aliquots of phosphate-buffered NBAF in screw-cap microfuge tubes outside the glove bag i. Loosen the caps ii. Incubate tubes in the glove bag for 24 hours prior to use c. Or take out 1 ml of NBAF from each tube to which you plan to transfer a mutant. 55. Transfer to NBAF pressure tubes with a 1 ml syringe. 56. Once your mutant grows in liquid, make duplicate freezer stocks as soon as possible.

Modifications for Geobacter metallireducens. Antibiotic stock Final concentration 30 mg/ml spectinomycin 300 µg/ml 500 mg/ml kanamycin 5 µg/ml in FW nitrate Aklujkar, Muktak 4-4

20 mg/ml kanamycin 200 µg/ml in Fe(III) citrate 2 mg/ml kanamycin 20 µg/ml in NBAF 200 µg/ml gentamicin 2 µg/ml in FW nitrate 10 mg/ml gentamicin 100 µg/ml in Fe(III) citrate 650 µg/ml gentamicin 6.5 µg/ml in NBAF