User:James Chappell/ Notes

Project Protocols

Protocols being used by specific projects

Papers on steady state

• The degradation rate of LuxR has been estimated by several experiments:
  • The LuxR was measured by western blot analyisis ~65minutes [1]
  • The LuxR rate of degradation is 0.7 hr-1 [2]

References

1. Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, and Givskov M. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology (Reading). 2002 Apr;148(Pt 4):1119-1127. DOI:10.1099/00221287-148-4-1119 |
2. Balagaddé FK, You L, Hansen CL, Arnold FH, and Quake SR. Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science. 2005 Jul 1;309(5731):137-40. DOI:10.1126/science.1109173 |
3. Ronen M, Rosenberg R, Shraiman BI, and Alon U. Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10555-60. DOI:10.1073/pnas.152046799 |

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1482501 http://www.che.caltech.edu/groups/fha/balagadde_supplemental.pdf http://www.duke.edu/~you/publications/You_nature2004.pdf http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed&cmd=Retrieve&list_uids=15159530

Optimizations

• Need to use a DNA extraction method that does not use ammonia. Recommend using sodium acetate rather than ammonia acetate to ethanol precipitate the plasmid.
• Standard protocol uses temperatures of 37^oC, however a shift to 24^oC can prolong for 20 hours and a 2 fold increase in protein synthesis

Mg²⁺ concentration is important. Optimum range is between 7-11mM.

• NaCl of greater than 50mM NaCl can inhibit translation.
• The level of DNA added should vary from 0.5ug to 4ug.

DNA Extraction

• Cultures to be purified grown for no longer than 16hours.
• Varying the elution buffer volume in the mini prep can vary the concentration and yield,

generally increasing the volume increases the yield but decreases the concentration.

• Host strains such as DH5alpha give good DNA extracts.

Protocol

Protocol:
Day 1: [show][hide]

1. Remove reagents from storage
2. Prepare AHL dilutions
3. Add cell extract in the appropriate wells following the plate schematic
4. Add the appropriate volume of purified DNA to the sample and wait for x minutes
5. Add AHL dilutions to appropriate well.
6. Vortex the cells gently and centrifuge 5 seconds to bring the reaction down to the bottom of the tube.

Preparation of Culture

1. Placed 5 ml of LB in a 15 ml tube
2. Added appropriate antibiotics into the tube
3. Picked a colony from the fresh overnight plate
4. Inoculated the colony in the LB media
5. Grew upto 16hours at 37°C in shaking incubator
   • 2x 15 Biobricks cloned

Miniprep

[show][hide]

1. Transfer 1.5 ml of bacterial cells to an eppendorf
2. Pellet cells by centrifuging for 30 seconds at maximum speed
3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to an eppendorf
4. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
5. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
6. Centrifuge for 10 min at maximum speed
7. Pipette the supernatant into the QIAprep spin column on top of the eppendorf
8. Centrifuge for 1 min and discard the flow-through.
9. Wash with 0.5 ml Buffer PB, centrifuge for 1 min, and discard the flow-through
   [Note: This step is unnecessary for cells without nucleases (DH5α, XL1-Blue]
10. Wash with 0.75 ml Buffer PE
11. Centrifuging for 1 min and discard the flow-through
12. Centrifuge for an additional 1 min to remove residual wash buffer
13. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube
14. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min