Team:Austin UTexas/Experiments
Contents
Experiments
From start to finish, a central goal of our project was burden quantification. Two experiments required for our progress were transformations and burden assays; select BioBrick parts in the iGEM registry were transformed into our burden monitor E. coli, and these strains were then moved to a 96 well plate to measure optical density and GFP fluorescence. This, along with our burden R script, then gave us the burden graphs seen in our Results. These two protocols are shown below.
Transformation
Though our parts and E. coli strain are specific to our project, these can be substituted with other plasmids and DNA relevant to any experiment.
Materials
- Competent cells
- Transforming DNA
- SOC medium
- Heat block or water bath
- Antibiotic plates
- Ice bucket with ice
Procedure
1. Take out a frozen tube of cells for each reaction, and appropriate controls.
2. Thaw tubes by holding them in your hand.
3. Add 0.5-1μL of the miniprepped plasmid (the transforming DNA) and incubate on ice for 30 minutes.
4. Transfer the tubes to a 42°C heat block or water bath for exactly 90 seconds.
5. Immediately transfer the tubes to ice for 1-2 minutes.
6. Add 450μL of SOC medium to each tube, then shake at 200-250RPM for 45 minutes.
7. Plate two different volumes (10μL and 50μL as an example) for each reaction on antibiotic plates.
Burden Assay
The iGEM registry's BioBricks were used as our plasmids-of-interest, but this protocol is generalized to allow for alternative plasmids.
Materials
- Transformed plasmids
- LB antibiotic plates
- LB medium
- 96 well plate
- Calibration strains
- Relevant antibiotics
- Plate reader
Procedure
Culture Preparation
1. With frozen stocks of plasmids-of-interest transformed into the burden monitor strain, streak LB plates with appropriate antibiotic. The maximum number of unique strains that can be run per assay is 23.
2. Streak the five calibration strains (JEB1204-1208) from frozen stocks onto LB-Chloramphenicol + Kanamycin plates.
3. Leave plates at 37°C overnight
4. Pick colonies and prepare overnight cultures of calibration strains into 5 mL LB-Chloramphenicol + Kanamycin as well as overnight cultures of each plasmid-of-interest into 5mL LB with appropriate antibiotic.
5. Place the bottle of LB to be used the next day into a 37°C incubator as well.
Plate Preparation
6. Begin prewarming the plate reader to 37°C before preparing the plate.
7. Vortex overnight cultures and place 5 µL into a 96-well plate in triplicate. Split the plate into 3 columns of equal width, so the triplicates will be in the same well position for each column. With 23 unique strains, leave wells A1, A4, A5, A8, A9, and A12 in addition to wells H1, H4, H5, H8, H9, and H12 for blanks. If less than 23 strains are being used, incorporate blanks in triplicate as needed. Fill blanks with 5 µL of room temperature LB. Make a 96-well-plate template representative of the experiment’s layout.
Figure 1. Well template of triplicates.
8. Take pre-warmed LB out of the incubator and use a multichannel pipette to load 195 µL of the warmed LB into each column. Gently pipette up and down 5 times to mix, being careful not to introduce bubbles, and place the lid.
Plate Reading
9. Load the burden assay method and start the plate reader. The method must run for at least 360 minutes.
10. Transform the 96-well-plate template into an electronic metadata layout as pictured here:
Figure 2. Example metadata in Excel.
11. Save the measurements generated as a csv.
Data Analysis
12. Use the metadata layout as well as the measurement files as inputs to the burden scripts in the Barrick Lab Github.