Lab 10

Analysis of P5, P5-33A and mutant promoter transformation and function

Goals for the Lab:

• Determine the relative strength of your self-designed promoter.
• Compare different promoter designs and find relationships between mutations and promoter strength.

The goal of this lab is for you to explore and discover the critical role of promoters in regulating transcription. Promoter strength can be defined as the rate at which RNA polymerase produces mRNA. To measure promoter strength directly, you would measure mRNA production, but that is technically challenging. An alternative is to measure mRNA production indirectly, by quantifying the amount of protein produced by translation of the mRNA. In general, the amount of protein produced is proportional to the quantity of mRNA present.

One way to quantify the amount of protein produced by the gene and thus, the strength of the promoter, is to take a digital photograph of colonies and then use the free software program lmageJ (http://rsb.info.nih.gov/ij/) to make measurements (Figure 1).

Part 1: Photograph and Categorize Colonies from Transformations

Materials

• plates with transformed bacteria from Lab 9
• plate with E. coli containing the standard (positive control) plasmid with the P5 promoter and amilCP Blue gene
• digital camera or phone with camera
• lab marker(s)
• UV light transilluminator to visualize and photograph GFP expression, and a white light box to enhance photography of AmilCP blue
• computers with ImageJ installed

Procedure

1. Photograph each of your transformations, making sure to include the labels. Avoid glare from overhead lights. To photograph the transformations, remove the lids and place the plates on the Safe light trans-illuminator open side down, to give the colonies maximum exposure to the blue light so that they will glow green to see the colonies that express GFP. Caution: The Safe light is extremely bright; photograph your colonies through the orange safety screen. When circling non-green GFP colonies use the orange protective glasses.
2. If you are photographing the plates on a white light trans-illuminator, remove the lids and place them open side up to reduce the glare from the plastic. If no white light trans-illuminator is available, plates containing pClone Blue may be photographed on white paper or using the white light box on the wall in lab. Note: our cell phone cameras leave brown lines on the pictures using the light box. Use a traditional digital camera found in lab to take these pictures.
3. Deduce the genotype of the plasmid inside the cells of each color colony. Review the background reading material to help you remember the genotypes for the different colony phenotypes.

Part 2: Measure Reporter Protein Production in E. coli Cells Using lmageJ: Quantifying Promoter Expression

To quantify with lmageJ, begin by opening an image file of a colony that was photographed as you were instructed. Use lmageJ to draw circles within the colonies or spots to be analyzed (you may have to zoom in first). The image is split into red, green, and blue channels, and a measurement is made by lmageJ of the intensity of color for each channel. Measurement of Blue chromoprotein output is obtained by dividing the blue channel intensity by the green channel intensity. The measurements obtained in the blue channel are divided by the measurements obtained in the green channel to correct for any difference in the red and blue channel measurements that result from the methodology used rather than from an actual difference in the amount of protein being expressed. For example, if a larger circle is drawn within colony A than within colony B, it may appear that the promoter in colony A is expressing more blue than the one in colony B, when in fact, the difference is simply a result of the size of the circle drawn. The lmageJ method of dividing blue channel measurements by green channel measurements has been validated by comparison to measurements using fluorometry.

Materials

• images of plates taken in Part 1
• free lmageJ software
• computer (Macintosh or Windows)

Procedure

You will import the image files from the photos of your plates into lmageJ and split the image into its red, green, and blue channels. You will then select a colony you want to measure and divide the value derived from measuring in the blue channel by that derived from measuring in the green channel. This ratio will provide a value for the strength of the promoter. The strength of the promoters you cloned using the GGA reaction (both the P5 and P533-A mutant promoter) will be compared to the standard P5 promoter.

1. Open the lmageJ software.

2. Drag and drop the files for the photographs of your plates onto lmageJ. Alternatively, under "File" click on "open." Find and select the image file of the bacterial plate with the yellowish white (non-green or non-red colonies) you want to open.

3. Zoom in on the image by using the magnifying glass or pushing the shift and + keys. Move the image by using the hand icon or by pressing the space bar and dragging with the mouse.

4. Under "Analyze" -> "Tools," open "ROI Manager" (ROI stands for Region of Interest). A separate window will open.

5. Click on an oval icon from the lmageJ toolbar and draw a circle inside a non-green colony with a mutant promoter in it. If you have blue colonies on your plate you should be measuring blue colonies. If not then you should choose a yellow/white colony. The circle does not have to be a perfect circle. Do not include any labels or other features that do not represent cell color.

6. Click on "Add (t)" in the ROI manager window. This step creates a numbered file associated with the ROI. The file numbers designate the coordinates for the ROI in the image and will appear in the ROI window.

7. In the lmageJ window, under "Image"> "Color," click on "Split Channels." This opens three additional windows labeled Red, Blue, and Green. The images will be referred to as the red, blue, and green channels. The software has divided the image into its red, blue, and green color components. Each of the new windows appears in black and white, although the intensity of the white seen in each image is proportional to the color intensity of the pixels at that location in the image.

8. Click on the blue channel. Your ROI circle is not visible at this point.

9. Under ROI Manager, select the numbered file for the ROI, and then click "Measure." Write down the Mean number without decimals, rounding up or down as appropriate (e.g., 219).

10. Click on the green channel image. In the ROI Manager, select the numbered file for the ROI and then click "Measure." Note: Your ROI circle is not visible at this point. Write down the Mean number (e.g., 135).

11. Divide the mean number from the blue channel by the mean number from the green channel to calculate the ratio that is the relative AmilCP blue expression measure (e.g,. 230/135 = 1.70). The level of AmilCP blue expression is an indication of the strength of your mutant promoter.

12. Repeat steps 2-11 to measure the AmilCP blue expression measure for 4 other colonies on your transformation plate to get a sense of the range of values.

13. Repeat steps 2-11 to determine the AmilCP blue expression measure of the P5 promoter and the P5-33A promoter.

14. a. You can compare the promoters by generating a ratio of the promoter strength of a given promoter to the strength of the standard P5 promoter in the positive control supplied to you by your instructor. To do this, divide the expression measure for both the cloned P5, P5-33A, and "SD" by the expression measure for the P5 standard promoter. These ratios will be called expression ratios.

b. Alternatively, you can directly divide the expression ratio for P5-33A and "SD" by the expression measure for the P5 promoter you cloned. The expression ratio gives the relative strength of your mutant promoter compared to the P5 you cloned.

15. Clean up your area as instructed. Record any observations in your notebook.

Part 3: Produce Representations of Data and Discuss Outcomes

1. Discuss your results with other students and your instructor. Focus on the purpose of these experiments to understand the role of promoters in gene regulation.
2. Think of effective ways to visually present your results in graphs or tables. A good visualization communicates key points easily with as few words as possible.
3. Finish your 5 minute in-class presentation with the data you collected today.
4. Present to the class.