20.109(S11):Prepare DNA for cloning (Day3)

Introduction

Last time you designed a modification to the P_lux-λ promoter in order to weaken its basal expression in the absence of AHL — you hope. To test your idea, we will have to make a new DNA construct, pED-IPTG-YFD, where YFD is "your favourite design." Depending on how modest a change you made, there could be multiple ways to implement it; for example, a few DNA basepairs can readily be mutated by a PCR-like approach called site-directed mutagenesis. In our case, we will clone a modified P_lux-λ insert into the pED-IPTG backbone. We are outsourcing insert construction to a company specializing in DNA synthesis. Recall that you designed two strands, each with a restriction site overhang complementary to the cognate one on the backbone. Before the strands can be ligated into the backbone, they must be annealed together into double-stranded DNA. This can be done by heating the DNA above its melting temperature (94-95 ° C) and then letting it slowly cool - very similar to how we denatured RNA and cooled it, except this time we want to strands to anneal rather than to form independent secondary structures.

The backbone must also be prepared for cloning, first by restriction digest, and then by gel purification. (What could happen if we didn't purify the large backbone fragment of interest from the rest of the digestion mixture?) As you will see today, one of the enzymes that we are using for cloning is not very good at cutting plasmids. (Lesson: always read all the way through the product notes!) Thus, we have prepared pED-IPTG-INS for you that was digested overnight with XmaI and BamHI. Because this plasmid is large (a little over 10 Kbp), you will run it on a relatively low weight percent gel (0.7 %) and use a kit especially designed for long DNA to purify it. While the gel runs, you will set up control digests to understand how they work and why we had to do this one for you.

Next time you will ligate the backbone and insert together, and then transform the new construct into bacteria. You will also continue gathering data about the 'broken' model system, namely the transfer function that maps IPTG concentration to Miller units. Combining this transfer function with the analogous one for the edge detector (that maps light to Miller units) will allow us to relate the IPTG- and light-sensitive systems to each other. In order to treat the different systems as consistently as possible, you will set up the cells carrying the transfer function plasmid for overnight culture from an OD of 0.0025, just like you did for the complete IPTG-sensitive system.

Now is also a good time to add to our understanding of P_lux-λ (specifically the lux part, as we covered λ last time) and POmpC. In doing so, we will learn about two fascinating pieces of prokaryote biology: two-component signaling systems and quorum sensing.

Bacteria don’t exactly have a reputation as great communicators! However, it turns out that these single-celled organisms do ‘talk’ to teach other in primitive ways. One thing that is useful for a bacterium to ‘hear’ about is how large a community it is living in: this is called quorum sensing, and can help with resource management in a population of bacteria. In Vibrio fischeri the quorum sensing system is composed of luxR and luxI - as you may by now have guessed. The autoinducer is AHL (made, indirectly, by luxI), while luxR is the induced gene that in turn activates transcription of luminescence genes such as necessary enzymes. The lux stands for light: V. fischeri glow (luminesce) when they reach a critical density, which happens only when they are growing in an associated host. [ADD REFERENCES]

In addition to having group-based responses (such as quorum sensing), individual bacteria often must respond to changes in their immediate environment without regard for what other nearby bacteria are doing. Just as quorum sensing is a system that is implemented in different ways in many different species of bacteria, so is two-component signaling (2CS) a common solution to a type of problem faced by diverse bacteria. The relevant 2CS system in the edge detector is osmoregulation: the membrane-spanning protein EnvZ detects changes in salt concentration, phosphorylates itself and then the responder protein OmpR, and finally the responder shuts off the ompC promoter. [FINISH AND ADD REFERENCES]

Protocols

Note that the protocol parts are staggered today due to several incubation steps, and thus the day will not be as long (I hope!) as it may seem.

Part 1: Run digested backbone on gel

1. Retrieve an aliquot of digested backbone, and add 2.5 μL of loading dye to it.
2. Load your sample onto the appropriate gel in the order listed below.
   • We are leaving every other well blank both for ease of cutting out bands and so you are not all squeezing onto one gel.
3. Once the gel starts running, continue with as many parts below as you can, and be sure to also pre-weigh an eppendorf tube for the purification.
4. When the gel stops running (45 min at 100 V), you will come and cut your bands out.

Gel 1:

Lane	Sample	Lane	Sample
1	-	6	DNA ladder (load 15 μL)
2	Red group	7	-
3	-	8	Yellow group
4	Orange group	9	-
5	-	10	-

Gel 2:

Lane	Sample	Lane	Sample
1	-	6	DNA ladder (load 15 μL)
2	Green group	7	-
3	-	8	Pink group
4	Blue group	9	-
5	-	10	Purple group

Part 2: Prepare short-term digests

Each lab team will prepare either a BamHI or an XmaI digest of the pED-IPTG-INS backbone. Although plasmid backbones must be cut with both enzymes before cloning (assuming two rather than one are used), single-enzyme digests are important controls to run. With a short insert, it is impossible to tell whether a plasmid has been cut once or twice, as either way the linear backbone will appear to be the same size.

The table below is for one reaction.

	Digest 1 (Red/Ylw/Blu/Prp)	Digest 2 (Org/Grn/Pnk)
Plasmid DNA	2.5 μL	2.5 μL
10X NEB buffer	2.5 μL of buffer#4	2.5 μL of buffer#4
Enzyme	5 U of XmaI (50,000 U/mL stock)	5 U of BamHI (20,000 U/mL stock)
H2O	For a total volume of 25 μL

1. Prepare a reaction cocktail for your assigned reaction above (digest 1 or digest 2) that includes water, buffer and enzyme. Prepare enough cocktail so that you pipet at least 1.5 μL of enzyme and then keep it on ice.
2. Combine 2.5 μL of the backbone with 22.5 μL of the cocktail, and move to 37°C for at least one [one-half??] hour.
   • While your samples are digesting, you can return to Parts XYZ of the protocol.

Part 3: Prepare transfer function cultures

decide on exact concentrations

The cells you will use today are strain ABXX, carrying plasmid pIPTG-lacZ.

1. If you are waiting for the spectrophotometer, you can begin by picking up X 14 mL round-bottom tubes and preparing sticky labels for your samples (described in step 6).
2. Measure the OD₆₀₀ value of a 1:10 dilution of your cells (use a total volume of 600 μL).
3. In a 50 mL conical tube, prepare enough LB with ampicillin (stock is 1000X) for X cultures.
4. Dilute the cell stock to an OD of 0.0025 in the 25 mL volume.
5. Distribute 2.5 mL of cells to each round-bottom tube with a 5 or 10 mL serological pipet.
6. Distribute additives to each tube such that you have duplicate samples with (0, 0.01, ... 5 mM) IPTG.
   • Recall that the IPTG stock is 1 M.
7. Place your tubes on the roller wheel, remembering the following good practices:
   • Completely snap shut the tube caps.
   • Be careful not to tip the tube "backwards" as you insert it in the roller wheel.
   • Balance your tubes as if on a centrifuge.
   • Turn the roller wheel back on!
8. Your cultures will be grown overnight, then moved to 4 °C until next time.

Part 4: Anneal oligos (if they have arrived)

1. Resuspend each oligo at 100 μM in DNase-free water.
2. Prepare a mixture that has each oligo at 25 μM in ligation buffer in a total volume of X &,u;L.
3. When everyone is ready, the mixtures to be annealed will be placed on a heat block at 94-95 °C for 5 minutes, then slowly cooled.
   • Because our heat blocks cool relatively quickly, the heat will be moved down in stages (10 min with the knob set at 70 °C, etc etc) before turning the block off.

Part 5: Purify digested backbone

1. As you did in Module 1, cut the relevant band out of the gel with a clean spatula, while wearing both safety glasses and a UV-protective face shield.
   • Keep your labmates aware of when the UV is on.
   • Remember to avoid cutting out excess gel that does not contain DNA. Working with more than 250 mg of gel will be difficult.
2. Get the weight of the gel slice by subtraction, then add the following: 3 volumes of buffer QX, and 2 volumes of nuclease-free water.
3. Vortex one of the QIAEX II bead aliquots for 30 seconds, then add 10 μL to your gel sample.
4. Incubate for 10 minutes in the 50 °C water bath, flicking every 2-3 min to help dissolve the gel.
   • If the color becomes blue or violet rather then yellow, see the teaching faculty. You will need to adjust the pH of your sample.
5. Centrifuge for 1 min at maximum speed, and pull off the supernatant. Either balance your tube against another group's or use a water-filled tube.
   • Throughout this protocol, you might initially use your P1000, then switch to the P200 to more easily pull off the last bit of liquid with a finer tip.
6. Add 500 μL of buffer QX, and flick the pellet to resuspend. Do not vortex, as this can shear long DNA fragments.
   • Resuspension by flicking is a but if an art, so feel free to ask the teaching faculty to demonstrate. With practice, you should be able to do it in about 30 seconds, not a few minutes.
   • One method that works well is flicking while holding the tube upside down, then smacking it on the lab bench to return the partially resuspended pellet to the bottom.
7. Centrifuge and remove the supernatant, then resuspend in 500 μL PE by flicking before centrifuging again.
8. Repeat the PE wash step. This time, be careful to remove all of the supernatant or your pellet will not dry in time. Because the pellet is fairly stable, when it is almost dry you can turn the tube upside down and tap it to expel any fine droplets (smaller than you can see) of ethanol. Again, you can ask the teaching faculty for assistance if needed.
9. The pellet should begin to turn white after 10 min, and be completely dry after 15-20 minutes.
   • Note that the thin top smear will never turn white; if you wait for that, you will over-dry your pellet.
10. Add 20 μL of TE buffer to the pellet and flick to resuspend. Due to the small volume, this resuspension step will take you longer to complete. Really tap the tube down hard on the bench to recover as much volume as possible (but do note centrifuge yet).
11. To best dissolve this long fragment of DNA, put your sample in the 50 °C bath for 10 minutes, or at least for 5 min.
12. Spin for 1 min, and then very carefully transfer the supernatant to a clean eppendorf tube. You should be able to recover 18-20 μL of fluid, but avoid the beads.
13. To make sure that you have not taken up any beads, or to get them out of the way if you have, briefly (3-5 seconds) centrifuge your tube and look for formation of a pellet.

Part 6: Run control digests on gel

Part 7: Measure backbone recovery

• You will need X μL of backbone for your control and experimental ligations. Being sure to reserve that much, measure the absorbance of your purified DNA.
  • Use a minimum volume of 400 μL in the UV micro-cuvettes.
  • To be above the detection limit of the spec., you will probably need to do <= a 1:X dilution.
  • Refer to the module 1 protocol if you are uncertain which absorbance readings you need to record.

For next time

Calculations for cloning

Some accountability re: learning RE cutting

Reagent list