21 pClone procedures

Day 1. Make dsDNA Promoters from ssDNA oligonucleotides

Oligo annealing goals:

You will anneal two pairs of partially complementary oligonucleotides together to form two promoters. You’ll clone these new promoters into the pClone Red plasmid in place of the original promoter.

Before you begin:

• Prepare a 1L beaker containing 500 mL boiling water. Cover with foil.
• Thaw all your reagents on ice.

1. Label two microcentrifuge tubes: P5 (the wildtype) and P5-33A (the mutant promoter).
2. Into each of the two tubes, pipette
   • 14 uL nuclease-free H20
   • 2 uL 10X annealing buffer (the annealing buffer is already made for you. It contains 1M NaCl and 100 mM Tris-HCl pH 7.4)
3. For tube P5, add 2 uL of P5-top and 2 uL of P5-bottom.
4. For tube P5-33A, add 2 uL of P5-33A-top and 2 uL of P5-33A-bottom.
5. Mix and spin down both tubes.
6. When all groups at your table are ready, place tubes in floating rack in a 1L beaker containing 500 mL boiling water. Keep the beaker covered with foil.
7. Boil for 5 minutes, turn off heat, leave tubes in water and, slowly cool for at least 2 hrs (preferably overnight)

Plate pouring

1. Wipe down your work surface with disinfectant.
2. Warm the ampicillin to room temperature.
3. Label the bottom of the plates around the edge.
4. Make sure agar is fully melted and that no lumps are present.
5. Allow the agar to cool to 55 ºC—the bottle can be held in a bare hand without pain, but it will still feel very warm. NOTE: If the agar is too hot, the heat will inactivate the antibiotic.
6. Add the antibiotic to indicated bottles (add 4 mL to 400 mL full bottle), swirl to mix. The final concentration of amplicillin is 100 ug/mL
7. Pour 6 LB-AMP plates per pair. These plates will be used to test your new promoters and negative control.
8. Each group of 4 needs to pour 1 LB plate to use as your ‘starter plate’ for transformations. This plate DOESN’T contain ampicillin since you’ll use it to grow E. coli that do NOT encode the gene for antibiotic resistance. Each group of 4 also needs to pour one more additional LB-AMP plate to grow a positive control RFP bacterial strain.
9. When pouring plates, add JUST ENOUGH agar so the bottom of the plate is covered (5 mm depth)
10. Replace the lid as soon as you add the agar.
11. After the agar has solidified, place the plates in a sleeve or zip loc bag and store them in the refrigerator. If there is condensation on the lids, leave them to dry overnight. There should be no liquid on the lids or surface when you plate bacteria.

Day 2: Golden Gate Assembly (GGA) Cloning of Promoters

In Part 2, you will use the cloning technique called “Golden Gate assembly” to replace the promoter in pClone Red. If the promoter is functional, this will change the protein made by the plasmid from GFP to RFP.

Dilute your promoters:

1. Prepare two microcentrifuge tubes: diluted P5 promoter and diluted P5-33A promoter.
2. Pipet 3 µL of the cooled annealed P5 oligonucleotides into the tube labeled “dilute P5 promoter,” and 3 µL of the cooled annealed P5-33A oligonucleotides into the tube labeled “dilute P5-33A promoter.”
3. Dilute the annealed oligonucleotide 100-fold by adding 297 µL of sterile deionized H20 to the 3 µL of the annealed P5 oligonucleotides and 297 µL of sterile distilled or deionized H20 to the annealed P5-33A oligonucleotides. Both promoters you are diluting should now have a volume of 300 µL.

Perform the Golden Gate Reaction

You will set up three reactions:

• P5
• P5-33A
• Negative control (C)

1. Prepare 3 PCR tubes: Label one tube “P5”, a second one “P5-33A”, and a third “- C” (for negative control). The control reaction will show you what happens when no promoter is available to be cloned. Also label the tubes in some way that distinguishes them as belonging to your group.
2. Thaw, mix, and spin down all master mix reagents except the enzymes. Keep all reagents on ice at all times.
3. Assemble the golden gate master mix on ice in a new 1.5 mL tube:

4. Pipet 8 µL of master mix to each of the tubes labeled “P5,” “P5-33A,” and “- C.”
5. Pipet 2 µL of your diluted dsDNA P5 promoter into the tube labeled “P5.”
6. Pipet 2 µL of your diluted dsDNA P5-33A promoter into the tube labeled “P5-33A.”
7. Pipet 2 µL of distilled or deionized water into the tube labeled “- C.”
8. The final volume in each of the tubes should be 10 µL

Run the following program in a thermocycler:

While the program runs, perform the paper activity to help you understand GGA cloning.

1. Cut out each piece surrounded by dotted lines.
2. The diagram of the plasmid shows you the key features. The large curved arrow indicates the promoter transcribing in the direction to express GFP.
3. The two DNA sequences in boxes on the sides are the oligos used to create the new promoter.
4. Assemble the dsDNA promoter P5 using the two ssDNA pieces of paper. Assemble the two single-stranded pieces of DNA so that the top strand appears 5’ to 3’ (left to right). Align the bases (use the rules of complementary basepairing) and leave four bases on each end to function as sticky ends. Tape these two strands together to form the new promoter you’re cloning into the plasmid.
5. Use scissors to perform the function of BsaI. Cut out the existing promoter in pClone Red (be careful~draw in pencil before you make the cut). Be careful to leave the sticky ends attached to the rest of the plasmid.
6. Insert your dsDNA promoter into pClone Red in place of the old promoter. Discuss with your partner: Is it possible to insert the P5 promoter backwards by rotating your paper 180 degrees? Why or why not?
7. Use clear tape to perform the function of DNA ligase and connect the P5 promoter to pClone Red.
8. Search the inserted promoter for the recognition sequence of BsaI. Could BsaI cut the new promoter out?
9. Through this paper activity, you removed the left-facing promoter that initiated transcription of GFP and replaced it with a right-facing promoter to initiate RFP transcription. This insertion required BsaI to cut and ligase to connect the new promoter.

Day 3. Transform ligations into E. coli and Spread Cells onto Plates

In this part of the lab, you will transform the ligated plasmids from the GGA reactions into MM294 E. coli and plate the bacteria on LB/amp plates. You will plate two different volumes of each transformation to ensure that you end up with colonies at a density you can distinguish and count. Each group will perform the following three transformations using the three GGA reactions from Day 2.

• P5 + pClone plasmid
• P5-33A + pClone plasmid
• pClone plasmid (negative control)

Note: your instructor will provide you with starter plate that contains MM294 bacteria grown in the absence of antibiotic (you poured this plate on Day 2). It’s important to use bacteria in the exponential stage of growth and so we plated the bacteria the day before the transformation lab. 

Important: Use sterile technique for the following steps:

1. Label one 1.5 mL transformation tube “P5,” one “P5-33A,” and the third “- C.”
2. Using sterile technique, add 250 µL of cold calcium chloride solution to each transformation tube. Use a new tip for each tube. Keep the tubes of calcium chloride on ice except during manipulations and when specifically instructed otherwise.
3. Use a sterile, plastic inoculating loop to transfer isolated colonies of E. coli from the starter plate to the tube labeled “P5.” The total area of the colonies picked should be 3-5 mm in diameter (no bigger than top of a pencil eraser).
   • Be careful not to transfer any agar from the plate along with the mass of cells.
   • Immerse the cells on the loop in the calcium chloride solution in the tube and vigorously spin the loop in the solution to dislodge the cell mass. Hold the tube up to the light to observe whether the cell mass has fallen off the loop.
4. Immediately, suspend the cells by repeatedly pipetting up and down, using a pipet with a clean, sterile tip. Examine the tube against the light to confirm that no visible clumps of cells remain in the tube or are lost in the pipet tip. The suspension should appear a milky white.
5. Immediately return the tube to ice.
6. Repeat steps 3-5 to add bacterial cells to the “P5-33A”and “- C” tubes. Use a new inoculating loop for each tube.
7. Pipet the entire volume of each of the P5, P5-33A, and negative control (- C) GGA reactions into the appropriately labeled tubes. After each addition, gently agitate the tube and return it to ice. Be sure to use a new pipet tip for each reaction.
8. Incubate all of the tubes on ice for 15 minutes.
9. While the tubes are incubating, label LB/amp plates as shown below. Label the base of the plate (not the lid), and keep your writing small and only around the perimeter of the plate.
   • P5+ pClone Red: 30 µL
   • P5+ pClone Red: 150 µL
   • P5-33A +pClone Red: 30 µL
   • P5-33A +pClone Red: 150 µL
   • (negative control) pClone Red: 30 µL
   • (negative control) pClone Red: 150 µL
10. Also include the name of the bacteria strain (MM294), the date, and some identifying initials to distinguish the plates as belonging to your group.
11. Following the 15-minute incubation on ice, “heat shock” the cells. Remove the tubes quickly from ice and immediately immerse them in a 42 °C water bath for 90 seconds. Gently agitate the tubes while they are in the water bath. Return the tubes directly to ice for 2 minutes. Note: In order for the protocol to work, movement of the tubes from the ice to the water bath must be done very quickly.
12. Use a pipet with a sterile tip to add 250 µL Luria broth (LB) to each tube. Gently tap the tubes with your finger to mix the LB with the cell suspension. Place the tubes in a rack or stand at room temperature for a 15-minute recovery.
13. Now you will remove some cells from each transformation tube and spread them on the plates. In order to make sure that you have a cell density that allows you to easily view and count the colonies, you will plate two different volumes of each transformation.
14. Pipet 30 µL of the P5 transformation onto the appropriately labeled plate.
15. Pipet 150 µL of the P5 transformation onto the appropriately labeled plates.
16. Use a sterile disposable spreader to spread the cell suspension on each plate. Use the same cell spreader to spread both the 30-µL and 150 -µL plates for each transformation, but spread the 30-µL plate first.
17. Repeat steps 14-16 plate the P5-33A and negative control transformations.
18. Incubate your plates in the 37 °C incubator overnight.
19. To evaluate the success of your experiment, streak a bacterial colony that expresses strong RFP fluorescence via a pLac promoter onto an LB-AMP plate.

Streak Plate Procedure:

1. Using sterile technique, use a loop to obtain a small amount of the RFP bacteria. Do not stab into the agar!
2. Partially open the cover of the LB-AMP plate and inoculate the first quadrant of the plate by gently streaking the surface of the medium. Use the edge of your loop to make your streak without damaging the surface of the plate. Remove the inoculating loop and resterilize it with a flame (or get a new loop).
3. Turn the plate 90°, insert the cool sterile loop and inoculate the second quadrant by streaking the surface of the plate at right angles to the original streak. As you carry out this procedure, be sure to cross your original streak 1-2 times so as to pick up inoculum.  Remove the loop and resterilize it (or get a new loop).
4. Turn the plate 90° and repeat the procedure to inoculate the third quadrant with a streak from the second quandrant.
5. Turn the plate again and repeat the procedure to inoculate the fourth quadrant with a streak from the third quandrant. Be sure this final streak pattern does not touch the original line of inoculum. Resterilize the loop.
6. Invert the plate, label it properly and incubate it at 37 °C.
7. Clean up your area and disinfect your workspace.

pClone Data Analysis. Count, Photograph, and Analyze Colonies

1. Photograph your positive control plate first to ensure RFP fluorescence is visible with the UV transilluminator.

2. Photograph each of your remaining plates, making sure to include the labels. Avoid glare from overhead lights. To photograph plates, remove the lids and place the plates on the UV light transilluminator open side down to give the colonies maximum exposure to the UV light so that they will glow more brightly. Caution: UV light can damage skin cell DNA and cause blindness. Make sure the lid is down before turning on the UV light.

4. Count the number of colonies of each color on each plate.
   1. If there are too many to count, you may divide the plate into pie-shaped wedges (four or eight parts, depending on how many colonies you have), count the number of colonies in one sector, and extrapolate that number to the entire number of colonies on the plate by multiplying the number of colonies counted by the number of wedges.
   2. If the colonies on the plate containing 150 µL are too crowded to count, use the plate(s) with 30 µL plated on them to collect your data. Count only one color at a time and use a lab marker to place a dot on top of each colony as you count it, so you don’t count a colony twice.
   3. Note: Green and yellowish white colonies may be hard to differentiate, depending upon how much GFP is being expressed. Use the photos taken on the UV light box to guide you in determining colony color.
5. Fill in the following Colony Results table using the data you collected. Remember that a single GGA condition may produce up to three different colony colors. Deduce the genotype of the plasmid inside the cells of each color.

5. Use the numbers in this table to calculate the efficiency with which you cloned your new promoters. For each GGA condition, divide the number of red fluorescent colonies by the total number of colonies and then multiply by 100. Perform the calculations using colony numbers from plates plated with the same volume of the transformation.

Efficiency = (Red colonies)/(Total colonies) *100

• Negative control:
• P5 promoter
• P5-33A promoter:

Measure reporter protein production in E. coli using ImageJ.

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 red channel by that derived from measuring in the green channel. This ratio will provide a value for the strength of the promoter at driving RFP expression. 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 Plac promoter found in the pClone Red Positive Control plasmid. The Plac promoter contains a different sequence than the P5 and P33A Promoters we are testing.

1. Download and then open the lmageJ software. For Mac users: The first time you run ImageJ, you may get a message: “ImageJ can’t be opened because it is from an unidentified developer” message, which can be bypassed by right clicking on ImageJ.app and selecting “Open” from the drop down menu.
2. Import an image by going to the “File” menu and clicking on “Open.” First, find and select the image file of the positive control bacterial plate with the RFP colonies.
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” à “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 single red (RFP+) 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. Add more colonies to your analysis (aim for 4 total in this analysis). To add the next colony, draw an oval on a different RFP+ colony and then click “Add (t)” to add this second colony to your analysis. Be consistent in what you are including in the ovals you draw for different colonies. Repeat until you’ve selected 4 RFP+ colonies.
8. When you are done, hit “SHOW ALL” in the ROI window to see all the colonies you are analyzing.
9. In the lmageJ window, under “Image” à “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.
10. Click on the red channel image. Your ROI circles are not visible at this point.
11. Under ROI Manager, select your first ROI file, and then click “Measure.” Write down the Mean number without decimals for your first colony, rounding up or down as needed.
12. Repeat this step for the remaining ROI files in your list and add your values to the table below in the “Red Channel” column.

13. Click on the green channel image. In the ROI Manager, click on the first ROI file and then click “Measure.” Write down the Mean number in the table above, which is the readout of the pixel intensity in the green channel.
14. Repeat for your remaining ROIs. Be careful that you match the correct green and red values to each colony.
15. For each ROI, in the table above, divide the mean number from the red channel by the mean number from the green channel to calculate the ratio that is the RFP expression measure. The level of RFP expression is an indication of the strength of the promoter.

Now you are ready to analyze your experimental plates and measure their promoter strength.

16. Repeat steps 2-15 to determine the RFP expression driven by the P5 promoter that you cloned using GGA.
17. The table below has rows for you to analyze 4 different RFP+ colonies from your P5 plate. If you don’t have 4 RFP+ colonies, fill in as much of it as you can.

18. Repeat steps 2-15 to determine the RFP expression driven by the P33A promoter that you cloned using GGA.

19. Compare the promoters by generating a ratio of the promoter strength of a given promoter to the strength of the Plac promoter in the positive control RFP bacterial strain. To do this, divide the normalized expression measure (Red/Green Means) for both the cloned P5 and P5-33A by the expression measure for the Plac standard promoter. These ratios will be called expression ratios.

20. Finally, you can directly divide the expression ratio for P5-33A 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 promoter that you cloned.
21. Make graphs to show your results. Make sure you include graphs to address: How efficiently did you generate RFP+ cells using these experimental promoters? Compare the strength of your two new promoters (P5 vs P33A) to our positive control. Include error bars on your graph!

Work on your pClone notebook. Here are tips on how to make sure you fully complete this assignment!