BestP: Measuring Fluorescence¶
Overview¶
The BestP experiment marks a shift from building DNA to measuring its activity. The goal is to assign a quantitative value—Relative Promoter Units (RPU)—to each promoter you identified in pP6. This allows us to compare their strengths under consistent conditions.
Why RPU?¶
Different experiments can yield different fluorescence values due to instrument settings, media conditions, or cell growth. To standardize promoter activity, we compare each sample’s fluorescence to a reference promoter: J23101, a commonly used medium-strength promoter from the Anderson library. Its activity is defined as 1 RPU.
Reference Plasmids¶
We use three plasmids from the Anderson promoter library:
- pJ12 – Contains J23112, a very weak promoter
- pJ01 – Contains J23101, the standard reference
- pJ19 – Contains J23119, a very strong promoter
Each has the same vector backbone and reporter gene (amilGFP) as your pP6 clones, ensuring fair comparison.
Experimental Workflow¶
You'll choose 4 pP6 clones to characterize—either your own from sequencing or others from the TPcon6B box. You'll also measure the reference plasmids above. Here’s the full procedure:
Step 1: Transformation¶
Transform all 7 plasmids (4 pP6 clones + pJ12, pJ01, pJ19) using a single aliquot of 100 μL competent cells. Use 16 μL of cells + KCM + 0.5 μL of plasmid DNA per transformation.
- Plate the transformations and label clearly with your assigned number
- Use carbencillin selection
- No rescue step is needed
Step 2: Picking¶
Pick 4 colonies per plasmid into a 24-well block:
- Add 4 mL of 2YT + carb to each well
- Pick in a left-to-right order to match data entry format
- Label with an airpore sheet
- Grow overnight in the multitron shaker
- Refrigerate plates and upload a photo (name it
BestP-XX)
Step 3: Measurement¶
Measure both fluorescence and OD₆₀₀ for all samples using a plate reader:
- Transfer 100 μL from each culture into a black-walled Tecan plate (2 technical replicates per sample)
- Use fluorescein settings to read amilGFP
- Save data to the USB stick
Step 4: Data Entry¶
Paste your raw OD and fluorescence readings into the provided spreadsheet. The sheet will:
- Calculate normalized fluorescence (per OD unit)
- Average technical and biological replicates
- Compute final RPU values with error bars
You’ll report these results to the main BestP Results sheet to help build a complete picture of the promoter library.
Example Results¶
| Promoter | RPU | Error |
|---|---|---|
| pJ01-C (J23101) | 1.00 | ±0.02 |
| 45C | 0.037 | ±0.003 |
| 7C | 0.679 | ±0.012 |
| 8A | 1.045 | ±0.019 |
Our ultimate goal with BestP is to measure the RPU of each pP6 clone and compare it to pJ19 to see if we have discovered a promoter with comparable or even greater activity. The strongest hits from this screen will move forward into the next stage of the project, where we’ll develop each into a part family. This is the final quality control step before we commit to that process.