Team:UT-Knoxville/Methods
<!DOCTYPE html>
Team:UT-Knoxville/Team
UTK
iGEM
Methods
Pu Promoter Characterization
- Seed cultures overnight with appropriate antibiotics in 37˚C, 200 RPM in a New Brunswick Scientific Excella E25 shaking incubator.
- In a 25 mL glass culture tube, add 10 ml fresh media such that OD=0.1.
- Allow to grow to mid-log phase, checking OD occasionally to find optimal induction time (~3 hours).
- For induction, add 0.1 mM IPTG and 1.0 mM m-xylene (or other aromatic). Cap with rubber stopper and aluminum seal to avoid losses to atmosphere.
- Time points were taken by extracting small volumes of culture from the tube with a syringe and needle. 150 µL aliquots were then measured for fluorescence (excitation 485/20, emission 528/20 nm) and optical density (OD600 nm) on a Biotech Synergy HT microplate reader over the course of 24 hours.
Note: M9 induction OD 600 may vary as maximum growth is less than LB, typically > 0.4.
First, frozen stock was used to inoculate 5 mL LB cultures of each strain and grown overnight at 37˚C, 200 RPM in a New Brunswick Scientific Excella E25 shaking incubator. The following morning, cultures were reseeded to OD=0.1 with 10 ml fresh M9 media in 25 mL glass culture tubes. Cells were grown to early/mid log phase, and then induced with 0.1 mM IPTG and 1.0 mM m-xylenes. Cultures were then capped with a rubber stopper and aluminum sealed to avoid atmospheric losses of volatile aromatics. Small samples were taken with a syringe and needle, and transferred to PCR tubes. Next, 150 µL of the sample was pipetted to a 96 well black, clear bottomed microplate for reading. Plates were shaken for 5 seconds, and measured for fluorescence (excitation 485/20, emission 528/20 nm) and optical density (OD600nm) on a Biotek Synergy HT microplate reader over the course of 24 hours.
Note: BBa_K2451013 was characterized in M9 media, as well as M9 with a tenth of the regular nitrogen content.
Liquid Benzaldehyde/Ampicillin Toxicity Test
- Seed cultures overnight with appropriate antibiotics in 37˚C, 200 RPM in a New Brunswick Scientific Excella E25 shaking incubator.
- Re-seed to OD=0.1 in 40 ml LB media in 500 ml flasks, and allow to grow to mid log phase at 37˚C, 200 RPM.
- Pour cultures into 50 ml Falcon tubes.
- Spin down at 4700 RPM, 22˚C for 5 minutes in a Thermo Scientific Sorvall Legend XTR benchtop centrifuge.
- Decant supernatant, resuspended in 1 mL sterile water. Take OD.
- Prepare media seed solutions: 200 mL LB + 0.5 mM IPTG + Benz/Amp.
- Distribute 10 mL of media into prelabeled 25 ml glass culture tues. Add 25 μg/mL chloramphenicol where necessary.
- Add high density cells from step 5 to tubes such that OD=0.1.
- Insert rubber seal and clamp aluminum seal.
- Take (t=0) OD measurement on a Thermo-Scientific Genysys30 Vis-spectrophotometer at wavelength 600 nm.
- Measure OD over the next 24 hours.
First, solid media was prepared by autoclaving a LB + 1% agar solution. The autoclaved media was cooled using a 50˚C water bath, after which 10 mM MgSO4 was added. The media was divided into 180 mL portions in 250-mL flasks, and we added ampicillin at various concentrations [3, 4.5, 6, 9 μg/mL]. 25 μg/mL chloramphenicol was also added to the media. The various media was mixed briefly in the 37˚C shaking incubator. The media was poured into miniature petri dishes, labeled, and stored at 4˚C for several days prior to the experiment.
To begin characterization, frozen stock was used to inoculate 5 mL LB cultures of each strain and grown overnight at 37˚C, 200 RPM in a New Brunswick Scientific Excella E25 shaking incubator. The following morning, the cultures were diluted to OD=1.0 with sterile 0.9% v/v NaCl water. OD=1.0 was diluted serially by factors of 10^2, 10^4, and 10^6. 5 μL droplet of each cell count to one quadrant of the plate via micropipette. The plates grew at 37˚C for 24 hours at which the pictures were taken.
