Lab 9

Assembling and Transforming the Promoters

NOTE: This will be a long lab - likely taking the entire 3.5 hour period. Please come to lab well prepared and understanding all of the steps of the protocols so that the procedures we need to do can be completed smoothly and on time.

Making dsDNA Promoters from ssDNA Oligonucleotides: Done by your instructors the day before lab 9

Materials

For each group:

• (3) tubes for PCR machine
• lab marker
• micropipets (2-20 μL)
• sterile tips for pipets
• container of ice
• wild-type pair of oligonucleotides (P5top and P5bottom)
• P5-33A top and bottom
• mutant pair of oligonucleotides designed by the group
• 10x annealing buffer
• sterile distilled or deionized water
• thermocycler

Procedure

In this part of the lab, you will anneal three pairs of partially complementary oligonucleotides together to form two promoters that can be cloned into pClone Blue in place of the plasmid's original promoter.

1. Label your PCR tubes "P5", "P5-33A", and "self-designed". Also label each tube in a way that will identify your group.

2. Into each tube, pipet 14 μL of sterile water and 2 μL of 10x annealing buffer (the annealing buffer contains 1 M NaCl and 100 mM Tris-HCI pH 7.4).

3. Pipet 2 μL of P5-top oligonucleotide and 2 μL of P5-bottom oligonucleotide into the microcentrifuge tube labeled "P5." Use a fresh pipet tip for each oligonucleotide. Gently mix the mixture using the second pipet tip. *Do not contaminate the common stock!*

4. Pipet 2 μL of P5-33A-top oligonucleotide and 2 μL of P5-33A-bottom oligonucleotide into the microcentrifuge tube labeled "P5-33A." Use a fresh pipet tip for each oligonucleotide. Gently mix the mixture using the second pipet tip. *Do not contaminate the common stock!*

5. Pipet 2 μL of your promoter-top oligonucleotide and 2 μL of your promoter-bottom oligonucleotide into the "self-designed" microcentrifuge tube labeled with your initials. Use a fresh pipet tip for each oligonucleotide. Gently mix the mixture using the second pipet tip.

6. The volume of each labeled tube should be 20 μL. Make sure all the liquid is at the bottom of each tube.

7. Place your labeled tubes into the thermocycler. The program will heat the tubes to 95C for 5 minutes, then slowly come back to room temperature. Leave the tubes in the thermocycler overnight.

Part 2: During Lab 9: Golden Gate Assembly (GGA) Cloning of Promoters

Materials

For each group:

• undiluted, annealed P5, P5-33A and team specific oligonucleotides on ice (if you will dilute your own oligonucleotides)
• 3 microcentrifuge tubes for diluting oligonucleotides
• 4 PCR tubes that will fit in the thermocycler
• micropipet, 0.5-10 μL
• micropipet, 100-1000 μL
• sterile tips for pipets
• sterile distilled or deionized water (700 μL)
• lab marker
• container for ice
• scissors
• clear tape
• paper GGA activity
• GGA Master Mix (on ice)

Shared by the class:

• thermal cycler

Procedure

In Part 2, you will use the cloning technique called "Golden Gate assembly" to replace the promoter in pClone Blue with the promoters you have created by annealing the oligonucleotides in Part 1. This will change the protein made by the plasmid from GFP to AmilCP blue .

Performing Golden Gate Assembly (GGA)

1. The tubes labeled in this step must be able to fit in the thermal cycler. Label four tubes "P5", "P5-33A," "SD", and "- 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 (e.g., use your initials).

2. Pipet 2 µL of your dsDNA promoters into each of the appropriate tubes.

3. Pipet 2 µL of distilled or deionized water into the tube labeled "- C."

4. Pipet 8 µL of master mix* to each of the tubes labeled "P5," "P5-33A", "your initials" and "- C." The final volume in each of the tubes should be 10 µL.

*The master mix contains all of the following reagents in the proportions shown:

• 2 µL of pClone Red or pClone Blue
• 1 µL 10x T4 DNA ligase buffer
• 0.5 µL Bsal
• 0.5 µL T4 DNA ligase
• 4 µL sterile distilled or deionized water

5. Place all four of your tubes in the thermal cycler. The thermal cycler is programmed as follows:

Stage 1

37°( for 1 minute (optimal temperature for Bsal)

16° ( for 1 minute (optimal temperature for DNA ligase)

These two steps are repeated for 45 cycles.

Stage 2

37°C for 15 minutes (final digestion with Bsal)

Stage 3

Hold at 4° C.

6. During the GGA cloning cycle, your instructor will distribute a paper activity that will help you understand what is happening at the molecular level during GGA. Use the following instructions for this activity.

a. Cut out each piece surrounded by dotted lines.

b. The diagram of the plasmid shows you the key features of the plasmid as it appears at the beginning of the reaction. The large curved arrow indicates the promoter transcribing in the direction to express GFP.

c. The two DNA sequences in boxes on the sides are the oligonucleotides used to create the new promoter.

d. 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). Make sure to align the bases and leave four bases on each end to function as single-stranded sticky ends. Tape these two strands together to form the new, dsDNA promoter you will clone into the plasmid. Refer to the figures on the sheet and in Lab 8 for help.

e. Use scissors to perform the function of Bsal. Cut out the existing promoter in pClone Blue (whichever plasmid you are using). Be very careful when you cut sticky ends; they must remain attached to the rest of the pClone Blue plasmid. Use a pencil to draw where you will cut before you start cutting.

f. Insert your dsDNA PS promoter into pClone Blue in place of the old promoter. Is it possible to insert the P5 promoter backward by rotating the paper 180 degrees? Explain your answer.

g. Use clear tape to perform the function of DNA ligase and connect the PS promoter to pClone Blue.

h. Search the inserted P5 promoter for the recognition sequence of Bsal (see Figure 3). Could Bsal cut the new promoter out of pClone Blue? Explain your answer.

i. On paper, you have removed the left-facing promoter that initiated transcription of GFP and replaced it with a right-facing promoter that will initiate transcription of AmilCP Blue. What you did on paper is the same thing that happens during GGA in the tube . At 37°C, Bsal cuts the DNA the way your scissors removed the old promoter. Ligase connects the new promoter by making covalent bonds (the clear tape) between the plasmid and the dsDNA promoter.

Part 3: Transform Ligations into E. coli and Spread Cells onto Plates of Selective Media

Materials

For each group:

• 4 GGA reactions from Part 2
• 4 transformation tubes
• DH5 alpha bacteria
• 3-mL vial of SOC
• 8 LB/carbinacillin plates
• bucket of ice
• rack or stand for transformation tubes
• waste container for material that has touched bacteria or medium
• waste container for other waste
• pipets for pipetting 10 μL, 30 μL, 250 μL, and 300 μL
• sterile tips for pipets

At a common workstation:

• sterile bacterial spreaders
• 42°C heat block
• 37 °C incubator (optional)
• Parafilm® or plastic wrap (optional)

Procedure

In this part of the lab, you will transform the ligated plasmids from the GGA reactions into DH5 alpha E. coli and spread the bacteria on LB/carb 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 four transformations using the four GGA reactions from Part 2:

• P5 + pClone plasmid

• P5 -33A+ pClone plasmid

• Self-designed oligo + pClone plasmid

• Negative control (water) +pClone plasmid

Throughout this protocol, work using sterile technique.

1. Label four transformation tubes of DH5 alpha cells "P5," "P5-33A," one "SD," and one "- C."

2. Immediately return the tube to ice.

3. Using an adjustable pipet and sterile pipet tips, pipet the entire volume of each of the four GGA reactions into the four 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.

4. Incubate all of the tubes on ice for 15-30 minutes.

8. While the tubes are incubating, label LB/carb plates as shown below.

• P5 + pClone Blue, 30 μL

• P5 + pClone Blue, 200 μL

• P5-33A + pClone Blue, 30 μL

• P5-33A + pClone Blue, 200 μL

• "SD" +pClone Blue, 30 μL

• "SD" +pClone Blue, 200 μL

• Negative control pClone Blue, 30 μL

• Negative control pClone Blue, 200 μL

Also include the name of the bacteria strain (DH5 alpha}, the date, and your initials to distinguish the plates as belonging to your group. Label each plate on the bottom surface as shown in Figure 6 to eliminate the possibility of any confusion due to misplacement of a lid.

9. Following the 15-30 minute incubation on ice, "heat shock" the cells. Remove the tubes quickly from ice and immediately immerse them in a 42°C heat block for 45 seconds. Return the tubes directly to ice for 5 minutes. Note: In order for the protocol to work, movement of the tubes from the ice to the heat block must be done very quickly.

10. Use a pipet with a sterile tip to add 500 μL SOC broth 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 37C for a 15-30 minute recovery.

11. 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.

a. Using a pipet and sterile tips, pipet 30 μL of each transformation onto the appropriately labeled plate.

b. Using a pipet and sterile tips, pipet 200 μL of each transformation onto the appropriately labeled plates .

c. 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 200-μL plates for each transformation, but spread the 30-μL plate first. Be sure to use a different spreader for the different transformations!

12. Incubate your plates as directed by your instructor.

13. Clean up your area as instructed. Record any observations or variations from the protocol that happened today.