SEPTEMBER

September 7

Two ligations completed, one with a 3:2 ration the other a 3:3 ratio of killer red and TRP.


Ligation Diagram

September 9

Ligation of the 3:3 ratio containing the Killer Red Digest and the psB1C3 Digest.

September 23

Digestion of Backbone again labeling the vial C2. Ran gel to see if the ligation was done correctly. It appeared that it was not run correctly; however the new ladder and dye being used did not show up because of its concentration.

September 28

Ligation between KR1 and C2 (backbone digest). Completed transformation via the Cloning a Gene into a Plasmid protocol.

September 30

Completed miniprep of KR Psb1.

OCTOBER

Wednesday, October 8

Sent miniprepped part for sequencing. Submitted KillerRed + Tryptophan BioBrick to iGEM headquarters.

Friday, October 10

Ran gel on miniprep of part to double check accuracy. Gel successful.

Saturday, October 11

Completed an experiment to develop a model for the part and provide proof that it works as expected.

Experimental Protocol

Experimental Protocol to Test KillerRed under control of tryptophan promoter in E. coli. Expose cells expressing KillerRed to white light for 0, 5, 10, 20, and 30 minutes. After exposure, perform a serial dilution to determine colony forming units so that we can determine the number of live cells in the culture.

To Prepare Before Experiment

• 6 – 250 mL flasks (autoclaved with foam stoppers and foil covering foam stopper)
• 9 packs of LB plates with Cm35
• 500 mL of LB, autoclaved
• 500 mL of M9 media
• Autoclave (1x M9 media: 5.64 g of M9 minimial salts and 500 mL of RO water)
• Autoclave (20% glucose: dissolve 10 g of glucose in 40 mL of water, bring volume to 50 mL)
• Autoclave (1.0 M MgSO4: make 25 mL of a 1 M solution)
• Autoclave (1.0 M CaCl2: make 25 mL of a 1 M solution)
• After all components are cooled: add 10 mL of the 20% glucose, 1 mL of 1 M MgSO4, and 50 uL of 1 M CaCl2 to the M9 media:
  • Filter Sterilize: 100 mM tryptophan (2 mL)
  • Make 4 ml of a 2.5 mg/mL stock solution of 3β-INDOLEACRYLIC ACID (IAA) in ethanol
  • Autoclave spreaders and glass beads
  • Make sure we have full boxes of tips especially p200 tips.

Day 1

Start 6 – 2 mL cultures of the E. coli containing KillerRed. Use LB and Cm35. Grow overnight at 37 ° C and 225 rpm.

Day 2

• Setup 3 flasks with 25 mL of M9 media and 1 mL of IAA stock solution (IAA did not come in in time for experiment), label flask, cover flasks in foil, label foil (same label)
• Setup 3 flasks with 25 mL of M9 media with 25 uL of 100 mM tryptophan, label flasks, cover flasks in foil, label foil
• Take an OD measurement of each culture (dilute each sample as follows: 100 uL of culture in 900 uL of water)
• Spin down 1 mL of culture in microcentrifuge tubes (max speed for 2 min). Remove liquid. Resuspend in 1 mL of M9 media without tryptophan. Keep cells in the dark at this point. Repeat spin down and resuspension to make sure all tryptophan in the media is removed.
• Inoculate each flask with approximately the same concentration of cells. (keep flasks in the dark) [C1V1 = C2V2]
• Grow cells at 37 ° Cand 225 rpm in the dark for 3-4 hours.
• While cells are growing, prepare for serial dilution part of the experiment.


• Pull plates out of refrigerator to allow them to come to room temperature
• Aliquot 900 uL of LB into microcentrifuges (need approximately 210 tubes) be as consistent as possible with your pipetting into each tube. (this is important)
• Label tubes (develop a short hand)
• Label plates

Experiment Time:

• 0 min timepoint: Take a 1 mL sample from each flask. Place on ice. Keep dark. [Alternatively if enough people are around, they can start with serial dilutions]
• Place flasks in Dr. Peebles lighted incubator set at 37 ° C and 225 rpm.
• After 5 min, 10 min, 20 min, and 30 min, take a 1 mL sample and place on ice. Keep samples in dark.
• For all collected samples, plate a serial dilution of the samples. See figure below. Try to work in the dark.
• Place all plates in the 37 ° C incubator. Can use bottom portion if needed. Probably should keep dark.

Figure 1, Visual for Serial Dilution

Day 3

• Remove plates from incubator.
• Count the plate that has approximately 100 cells on it. [if there is not enough time to count all cells, than store at 4 ° C and count at a later date]
• If you choose a plate with too few cells, one would expect too much variation in the number.
• If you choose a plate with too many cells, it is hard to count.
• Tip: use a sharpie to mark each cell that is counted. This will help reduce double counting cells

While our experiment did show what looked like a kill curve similar to what might be expected, the cells appeared to bounce back relatively quickly and the perceived difference between our samples and our controls (controls contained Trp which should have repressed KillerRed) was not large enough to yield conclusive data. We believe that this is because the cells expressed their own Trp after a few minutes, causing KillerRed to be repressed. This problem would be solved by adding IAA which we received the next week.

Results of Experiment

Wednesday, October 15

To save time and resources, the same protocol was followed using left over M9 media with only two samples per time point and only tested at 0, 15 and 30 minutes. We also added IAA to the samples without Trp added, so any Trp expressed by the cells would not interfere with KillerRed function.

Previous